We especially want to thank the members of the Gerber lab for technical instructions on their experimental setup and comments on the manuscript. We also thank Lyubov Pankevych for fly care and maintenance of the wild type CantonS stock. This work is supported by the DFG grant TH1584/1-1, the SNF grant 31003A_132812/1 and the Zukunftskolleg of the University of Konstanz (all to AST).

1. Preparation
<ol>
	<li>Drosophila wild-type larvae are raised at 25 &deg;C and 60%-80% humidity in a 14/10 light/dark cycle. For controlling the exact age of the larvae always 20 females are put with 10 males into one vial (6 cm height and 2.5 cm diameter) that includes about 6 ml of standard fly food. Flies are allowed to lay eggs for 12 hr and are transferred to a new vial on the second day. 5-6 days after egg laying larvae reach the feeding 3rd instar stage if raised at 25 &deg;C and can now be used for the behavioral experiment. However, one has to ensure to only take larvae that are still in the food and not the larvae from the side of the vial. These larvae have already reached "wandering 3rd instar stage" - shortly before pupation - and their usage complicates the interpretation of the results.</li>
	<li>Preparation of 2.5% agarose Petri dishes (other labs use also agar concentrations of 1% throughout the experiment, however lower concentrations may allow the feeding 3rd instar larvae to dig into the substrate): Dissolve 2.5 g agarose in 100 ml ddH2O. Heat up the solution in a microwave until it starts boiling. Carefully agitate the solution and put it back into the microwave until all agarose is dissolved. Pour the hot agarose solution into Petri dishes such that the bottom of the Petri dishes is completely covered and the agarose solution forms a smooth surface. Let the solution cool down to room temperature and close the lids. Do not immediately close the lids, because it would allow for condensation of water at the lids.</li>
	<li>Preparation of 2M fructose Petri dishes: Dissolve 2.5 g agarose in 100 ml ddH2O (again, usage of 1% agar concentration is possible; however it may allow feeding 3rd instar larvae to dig into the substrate). Heat up the solution in a microwave until it starts boiling. Carefully agitate the solution and put it back into the microwave until all agarose is dissolved. Carefully add 35 g of fructose into the hot solution; slowly agitate the mixture until the sugar is dissolved to avoid boiling retardation. Pour the hot fructose-agarose solution into Petri dishes such that the bottom is completely covered and the fructose-agarose solution forms a smooth surface. Let the solution cool down to room temperature and close the lids. Do not immediately close the lids, because it would allow for condensation of water at the lids.</li>
	<li>Preparation of 1-octanol (OCT) odor containers: Fill 10 &mu;l of pure OCT into a custom made Teflon odor container and close it with a lid that has several small holes to allow for evaporation of the odor. A detailed description of the containers is given in Gerber and Stocker 2007. Prepare three odor containers for OCT. Odor containers allow for the evaporation of the inserted chemicals, but avoid that larvae can directly contact them. Thus, the here described experiments specifically address olfactory learning in larvae without perturbing gustatory side effects.</li>
	<li>Preparation of amylacetate (AM) odor containers: Dilute AM 1:50 in paraffin oil. Fill 10 &mu;l of the dilution into a custom made Teflon odor container and close it with a lid that has several small holes to allow for evaporation of the odor. Prepare three odor containers for AM. The dilution is important for practical reasons, namely to avoid a strong preference for one odor over the other that may mask a learning-dependent change in the relative preference between the two odors. The equal attraction for both odors may need to be confirmed in the lab before the experiment by applying the later described test (2.5) with na&iuml;ve animals. The values here presented are based on several publications of the Gerber lab that were recently reproduced by our lab9,15,16.</li>
	<li>Labeling of the Petri dishes: Before the behavioral experiments all Petri dishes have to be coded. That means that fructose containing Petri dishes have to be marked for example with an "X" or an "A" and agarose only Petri dishes with a "Y" or a "B". This code should be revealed to the experimenter only after all data have been recorded. By performing the experiments "blind" it is thus not possible that the expectations of the experimenter can affect the performance of the larvae. To ease an understanding for the broad readership in the following we will only talk about the odors as conditioned stimuli (CS1 or CS2) that are either rewarded when presented on a fructose Petri dish(+) or non-rewarded when presented on an agarose only Petri dish(-).</li>
</ol>
2. Sugar Reward Training and Test
<ol>
	<li>Collect 30 feeding 3rd instar larvae from a food vial. Transfer them to a first Petri dish that contains some drops of tap water and carefully move them forwards and backwards with a brush. Transfer them to a second Petri dish that contains also some drops of tap water to check that no food paste remains on the bodywall of the larvae; otherwise larvae would be able to experience the food odor during the experiment. This would likely obscure the learning process and their performance in the test situation.</li>
	<li>Training: To train larvae to associate odors with an appetitive sugar cue the following regime is applied. Put an OCT odor container on the left and right side of an "X" marked - thus, fructose reward containing (+) Petri dish ("blind" experiment, for details see 1.6). Put the group of 30 feeding 3rd instar larvae onto the middle of the Petri dish, close the lid and wait for 5 min while the animals are exposed to OCT. Make sure that the larvae are not trapped inside the water drop and can overcome the surface tension of it. By that larvae can freely move on the Petri dish and experience olfactory and/or gustatory stimuli.</li>
	<li>Training: Remove the larvae from the Petri dish with a moistened brush and transfer them onto a second Petri dish that is labeled with a "Y" - thus, agarose only (-) containing Petri dish - and has an AM odor container located on its left and right side. Close the lid and wait for 5 min while the animals are exposed to AM.</li>
	<li>Training: Repeat 2.2) and 2.3) twice, such that all 30 larvae experience three training cycles: CS1 / (+) - CS2 / (-); CS1 / (+) - CS2 / (-); CS1 / (+) - CS2 / (-). In this experiment CS1 represents OCT and CS2 codes for AM.</li>
	<li>Test: Place one AM and one OCT odor container on the opposing sites of an agarose-only Petri dish. Transfer the trained animals to the middle of the test Petri dish. Close the lid and wait for 5 min. Subsequently count the number of larvae on the left side, the middle and right side of the test Petri dish.</li>
	<li>Repeat steps 2.1) to 2.5) with a second group of 30 feeding 3rd instar larvae but swap the experimental roles of AM and OCT such that the animals receive the following training: CS2 / (+) - CS1 / (-); CS2 / (+) - CS1 / (-); CS2 / (+) - CS1 / (-). In this experiment CS1 represents OCT and CS2 codes for AM.</li>
	<li>Possible arrangements for the training. Above we present a training that exists of three training trials of either OCT / (+) - AM / (-) or in the reciprocal group of also three training trials of AM / (+) - OCT / (-). However to avoid sequence dependent effects during the training it is important to vary the sequence of the stimuli in following repetitions of the complete experiment. By varying the CS1 or CS2 order and also the reward presentation in the first or second presented plate, four different sequences for the training trials are possible:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>First group</td>
			<td>CS1 / (+) - CS2 / (-)</td>
			<td>Reciprocal group</td>
			<td>CS2 / (+) - CS1 / (-)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>CS1 / (-) - CS2 / (+)</td>
			<td>&nbsp;</td>
			<td>CS2 / (-) - CS1 / (+)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>CS2 / (+) - CS2 / (-)</td>
			<td>&nbsp;</td>
			<td>CS1 / (+) - CS2 / (-)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>CS2 / (-) - CS1 / (+)</td>
			<td>&nbsp;</td>
			<td>CS1 / (-) - CS2 / (+)</td>
		</tr>
	</tbody>
</table>
To prevent systematic effects of stimuli in the surrounding experimental environment, one should perform the test in one-half of the cases such that OCT is presented on the left and AM to the right. In the other half of the cases AM should be presented on the left and OCT on the right.
3. Tests for Task-relevant Sensory-motor Faculties
The design of the above described experiments allows for analyzing odor-sugar learning in wild type feeding 3rd instar larvae on its own. However, in daily lab life researchers usually use two or more different experimental groups of larvae to compare, if olfactory learning depends on a particular gene, a specific set of neurons, a mutant stock, a special food diet, different rearing conditions, toxic chemicals added during development, etc. Thus, in all cases when two or more experimental groups of larvae are tested one has to do a set of mandatory control experiments to test, if the different groups of larvae show proper sensory-motor acuities. This becomes mandatory as potential phenotypes are not necessarily due to reduced or abolished abilities to associate odors with sugar. Rather, potential learning defects could be based on defects at any step of the sensory-motor circuitry in the processing of odors and/or sugar. Or in other words, if a mutant larva is not able to sense sugar, it cannot establish a sugar memory. But this does not allow concluding that the larva cannot learn. In detail the following control experiments have to be done to test for proper OCT, AM and fructose processing of transgenic larvae.
1. Test for na&iuml;ve OCT preference
Collect 30 feeding 3rd instar larvae from a food vial. Wash them carefully in tap water as described in 2.1. Put a single OCT odor container on one side of an agarose Petri dish, add the larvae onto the middle of the Petri dish, close the lid and wait for 5 min, such that the larvae can crawl on the Petri dish and orient towards the OCT odor source. Subsequently count the number of larvae on the left side, in the middle and on the right side of the test Petri dish.
2. Test for na&iuml;ve AM preference
Collect 30 feeding 3rd instar larvae from a food vial. Wash them carefully in tap water as described in 2.1. Put a single AM odor container on one side of an agarose Petri dish, add the larvae onto the middle of the Petri dish, close the lid and wait for 5 min, such that the larvae can crawl on the Petri dish and orient towards the AM odor source. Subsequently count the number of larvae on the left side, in the middle and on the right side of the test Petri dish.
3. Test for na&iuml;ve sugar preference
Collect 30 feeding 3rd instar larvae from a food vial. Wash them carefully in tap water as described in 2.1. Prepare Petri dishes that contain 2.5% agarose in one half and a 2M fructose-agarose mixture in the other half. Add the larvae onto the Petri dish, close the lid and wait for 5 min, such that the larvae can crawl on the Petri dish and orient towards the fructose containing side. Subsequently count the number of larvae on the left side, in the middle and on the right side of the test Petri dish.
Preparation of half-half Petri dishes: Prepare normal agarose plates as described above in section 1.2. When the agarose filled Petri dishes are cooled down, carefully cut the agarose along the vertical axis with a scalpel. Remove one half of the agarose from the Petri dish. Add a hot fructose-agarose solution (for preparation see 1.3) to the empty half of the Petri dish. Be careful that both halves match and do not form a defined edge - this affects the larval choice behavior and renders a behavioral analysis rather difficult.4.
Sham training
Despite testing if transgenic feeding 3rd instar larvae are able to distinguish on a wild type level between OCT and air (3.1), AM and air (3.2) and sugar and pure agarose (3.3), an additional set of test experiments has recently been introduced (for discussion see Gerber and Stocker, 2007). The rationale for these experiments is the following. During the training larvae undergo massive handling and successive odor and sugar stimulation. Thus, it is well possible that the observed learning phenotype is misleading (although na&iuml;ve odor and sugar perception tests are on a wild type level!). Indeed, it is possible that the transgenic animals differ from wild type larvae with respect to stress resistance, motivation, fatigue, sensory adaptation, contextual learning, and changes in satiety. Thus, Michels et al. (2005) introduced controls that test whether a given mutant is able to (1) detect AM versus an empty odor container if you treat the larvae exactly as during training except that you omit the reward and merely expose to both odors; (2) detect OCT after the same regime; (3) detect AM versus an empty odor container, if you treat the larvae in a training-like way except that you omit the odors and merely expose to the reward; and (4) detect OCT after the same regimen. For a comprehensive discussion and further details on the methods please see Michels et al (2005) and Gerber and Stocker (2007).
4. Data Analysis for Sugar Reward Learning
<ol>
	<li>To evaluate the data of the sugar reward learning protocol calculate a OCT preference index (PREF OCT) for each of the two reciprocally trained groups: 
	For the first group that received OCT/(+) - AM/ (-) training: 
	PREF OCT (OCT+/AM-) = (# of larvae on OCT side - # of larvae on AM side) / # of all larvae within the left, right and middle zones 
	For the second group that received AM/ (+) - OCT/(-) training: 
	PREF AM (AM+/OCT-) = (# of larvae on AM side - # of larvae on OCT side) / # of all larvae within the left, right and middle zones</li>
</ol>
<ol start="2">
	<li>Calculate a performance index (PI) for the two PREF values from 4.1). The PI represents associative learning by cancelling out perturbing effects of odor and punishment exposure, passage of time and handling: 
	PI = (PREF OCT (OCT+/AM) + PREF AM (AM+/OCT)) / 2 
	Thus PIs can range from -1 to 1. Significantly negative values represent aversive learning, whereas significantly positive values describe appetitive learning. A complete experiment usually comprises of 10 or more PIs. Data are visualized as box plots including all values of a given experimental group. 50% of the values being located within the box; the median performance index is indicated as a bold line within the box plot.</li>
</ol>
5. Data Analysis for Task-relevant Sensory-motor Faculties
<ol>
	<li>To evaluate the data when testing for proper OCT odor processing calculate an OCT odor preference index as follows: 
	Odor PREF OCT = (# of larvae on OCT side - # of larvae on the other side) / # of all larvae within the left, right and middle zones</li>
	<li>To evaluate the data when testing for proper AM odor perception calculate a AM odor preference index as follows: 
	Odor PREF AM = (# of larvae on AM side - # of larvae on the other side) / # of all larvae within the left, right and middle zones</li>
	<li>To evaluate the data when testing for proper fructose perception calculate a fructose preference index as follows: 
	PREF fructose = (# of larvae on fructose side - # of larvae on the other side) / # of all larvae within the left, right and middle zones</li>
	<li>Details for the sham training are given in Michels et al. 2005.</li>
</ol>

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Biol. 16, 1741-1747 (2006).</li><li>Rosenzweig, M., 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+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li><li>Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+electrical+properties+in+Drosophila+neurons+developing+without+synaptic+transmission.">Altered electrical properties in Drosophila neurons developing without synaptic transmission.</a> J. Neurosci. 21, 1523-1531 (2001).</li><li>Chen, Y. C., Mishra, D., Schmitt, L., Schmuker, M., Gerber, 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+behavioral+odor+similarity+"space"+in+larval+Drosophila.">A behavioral odor similarity "space" in larval Drosophila.</a> Chem. Senses. 36, 237-249 (2011).</li><li>Saumweber, T., Husse, J., Gerber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+attractiveness+and+associative+learnability+of+odors+can+be+dissociated+in+larval+Drosophila.">Innate attractiveness and associative learnability of odors can be dissociated in larval Drosophila.</a> Chem. Senses. 36, 223-235 (2011).</li><li>von Essen, A. M., Pauls, D., Thum, A. S., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+visual+classical+conditioning+in+Drosophila+larvae.">Capacity of visual classical conditioning in Drosophila larvae.</a> Behav. Neurosci. , (2011).</li><li>Honjo, K., Furukubo-Tokunaga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cAMP+response+element-binding+protein-dependent+medium-term+memory+by+appetitive+gustatory+reinforcement+in+Drosophila+larvae.">Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae.</a> J. Neurosci. 25, 7905-7913 (2005).</li><li>Honjo, K., Furukubo-Tokunaga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+neuronal+networks+and+biochemical+pathways+for+appetitive+and+aversive+memory+in+Drosophila+larvae.">Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae.</a> J. Neurosci. 29, 852-862 (2009).</li><li>Khurana, 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=Olfactory+Conditioning+in+the+Third+Instar+Larvae+of+Drosophila+melanogaster+Using+Heat+Shock+Reinforcement.">Olfactory Conditioning in the Third Instar Larvae of Drosophila melanogaster Using Heat Shock Reinforcement.</a> Behav. Genet. 42, 151-161 (2012).</li><li>Tully, T., Cambiazo, V., Kruse, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+through+metamorphosis+in+normal+and+mutant.">Memory through metamorphosis in normal and mutant.</a> 14, 68-74 (1994).</li><li>Michels, 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=Cellular+site+and+molecular+mode+of+synapsin+action+in+associative+learning.">Cellular site and molecular mode of synapsin action in associative learning.</a> Learn Mem. 18, 332-344 (2011).</li><li>Saumweber, T., 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=Behavioral+and+synaptic+plasticity+are+impaired+upon+lack+of+the+synaptic+protein+SAP47.">Behavioral and synaptic plasticity are impaired upon lack of the synaptic protein SAP47.</a> J. Neurosci. 31, 3508-3518 (2011).</li><li>Pfeiffer, B. 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=Refinement+of+tools+for+targeted+gene+expression+in+Drosophila.">Refinement of tools for targeted gene expression in Drosophila.</a> Genetics. 186, 735-755 (2010).</li><li>Rosenzweig, M., Kang, K., Garrity, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+TRP+channels+are+required+for+warm+and+cool+avoidance+in+Drosophila+melanogaster.">Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 14668-14673 (2008).</li></ol>

No conflicts of interest declared.

The described setup in Drosophila larvae allows for the investigation of associative olfactory learning within a comparably elementary brain. The approach is simple, cheap, easy to establish in a lab and does not require high-tech equipment9. We present a version of the experiment, to study appetitive associative learning reinforced by fructose reward11. The described setup is based on a series of parametrical studies that comprehensively investigated variations in the number of trai...

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 <td>Company</td>
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 <td>Fructose </td>
 <td>Sigma </td>
 <td>47740</td>
 <td>57-48-7</td>
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 <td>NaCl</td>
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 <td>Agarose</td>
 <td>Sigma</td>
 <td>A5093</td>
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 <td>1-octanol</td>
 <td>Sigma</td>
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 <td>Amylacetate</td>
 <td>Sigma</td>
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</table>

appetitive associative olfactory learning in drosophila larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Figure 1A shows an overview of the experimental procedures for larval olfactory associative learning. By pairing one of the two presented odors with a sugar reward larvae acquire the behavior potential to express an attractive response towards the rewarded odor in comparison to the unrewarded odor. Two groups of larvae are always trained by either pairing the reinforcer with the odor OCT or AM. The performance index (PI) measures the associative function as the difference in preference between the...

Watch this Scientific Journal Video about Appetitive Associative Olfactory Learning in Drosophila Larvae at JoVE.com

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

appetitive-associative-olfactory-learning-in-drosophila-larvae

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18.8K Views.  University of Konstanz. Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Video: Appetitive Associative Olfactory Learning in Drosophila Larvae

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.