Name: drosophila adult olfactory shock learning
Uploaded: 2014-08-07T19:45:00
Duration: 9 min 48 s
Description: Watch this Scientific Journal Video about Drosophila Adult Olfactory Shock Learning at JoVE.com

We acknowledge Bloomington stock centers for the fly strain. This work was supported by research grant from BBSRC (BB/G008973/1).

1. Fly Preparation
<ol>
	<li>Outcross all mutants, Gal4/UAS and other lines with a wildtype strain, such as CSw-, for at least six generations prior to the behavioral experiments to control for genetic background26.</li>
	<li>Grow flies on a standard cornmeal, yeast and molasses food diet under a 12:12 hr light-dark cycle at 25 &#176;C unless specific manipulations require a different temperature.
	<ol>
		<li>To determine the effects of transgene expression: Use 18 &#176;C to prevent the expression of transgenes in Gal4 Gal80ts (TARGET system) through development and then move the flies to a 30 &#176;C incubator 1-2 days before the behavioral experiment. Conduct the experiment at 30 &#176;C to determine the effects of transgene expression3,4,6,7,14.</li>
		<li>For experiments using heat-activated TRPA1 channels to stimulate neurons: Raise the flies at 23 &#176;C, which is a temperature known to maintain an inactive channel, and then shift to a behavior room at 30 &#176;C to activate the TRPA1 expressing neurons.</li>
		<li>For experiments using Shibiretemperature sensitive to block synaptic output11,14,24: Rear the flies at 18 &#176;C and conduct testing at 30 &#176;C.</li>
	</ol>
	</li>
	<li>Collect the flies 1-2 days before the experiment and count them in groups of approximately 25 under light CO2 anesthesia. Store the flies at least O/N in food vials (without yeast) at 25 &#176;C (unless specific manipulations required a different temperature) and 70% relative humidity in an environmentally controlled room with 12:12 hr light:dark conditions until the time of the experiment. 
	Note: This storage allows the flies to acclimatize to the subsequent learning test performed in the environmentally controlled room, which had the optimal conditions for Drosophila learning and, importantly, removes any daily environmental variations that might have affected the behavioral phenotype.</li>
</ol>
2. Preparation Before the Experiment
<ol>
	<li>Perform the experiments in a custom-made Perspex T-maze (Figure 1).</li>
	<li>Regularly check the tube fitting to ensure that an airtight seal is obtained during the experiment. If necessary, change the O-rings that seal the inside compartments of the T-maze.</li>
	<li>Place custom-made copper grids inside the training tubes. Check and clean these grids regularly, and replace if oxidized. Attach the copper grids to wires via crocodile clips that run to a switch-box connected to an electric stimulator. Use a voltmeter to ensure that the apparatus is delivering the required shock.</li>
	<li>Use g-clamps to hold the maze tightly to avoid any air leakage.</li>
	<li>Attach the T-maze to tubing that runs to an air pump, to allow odors to be drawn across the flies and subsequently removed from the T-maze. Maintain mild airflow at ~2&#160;L/min</li>
</ol>
3. Odor Dilutions
<ol>
	<li>Use two different odors at concentrations such that the flies show an equal preference for both odors. Use 4-methylcyclohexanol (1:67) and 3-octanol (1:100) diluted in mineral oil7,13. 
	Note: Carefully determine these concentrations, which will vary by laboratory. For instance others use 1:10 for both odors24 . Other commonly used odors include ethyl acetate and iso-amyl acetate.</li>
	<li>Pipette 30 &#956;l&#160;of the diluted odor into a custom-made odor cup placed in an odor block covered by a plastic tube with a perforated top that allows air to be drawn over the odor in the cup, thus exposing the flies to an odor plume.</li>
</ol>
4. Training Protocol (Figures 1 and 2)
<ol>
	<li>For adult olfactory shock conditioning, perform all experiments under a dim red light (i.e., red LED), which allows the researcher to see but prevents the fly from seeing, thus allowing the flies to concentrate on olfaction as opposed to visual inputs.</li>
	<li>Introduce the flies into the training tube and then&#160;attach&#160;to the T-maze and allow them to adapt to the tube and airflow for 90 sec.</li>
	<li>Present the first odor (4-methylcyclohexanol, MCH) with a 60-V shock (consisting of twelve 1.25&#160;sec pulses with 3.75&#160;sec inter-pulse intervals) for a total duration of 60 sec.</li>
	<li>Follow the shock with a 30&#160;sec rest period without an odor or shock.</li>
	<li>Present the second odor (3-octanol, OCT) for 60 sec without a shock.</li>
	<li>Follow the shock with a 30&#160;sec rest period without an odor or shock.</li>
	<li>Move the flies from the training chamber into the central chamber of the T-maze by turning the T-maze on its side and gently banging the bottom of the T-maze on a soft surface such as an old mouse mat. Maintain the flies in the central chamber for 90 sec.</li>
	<li>Fit the choice tubes into the bottom of the apparatus to form the T-maze.</li>
	<li>To measure learning, move the flies to the choice point of the T-maze, where they were simultaneously exposed to both odors and move towards one. Conduct a test period for 120 sec.</li>
	<li>Trap the flies in the choice tubes by sliding the central chamber up thereby blocking the ends of the choice tubes. Collect the flies in each arm of the T-maze and in the central compartment into food vials and count.</li>
	<li>To measure memory, collect the flies after training (4.6) and transfer them from the T-maze to food vials without yeast. Store flies in the dark at 25 &#176;C and 70% humidity for the remaining time necessary to determine the memory phase of interest (see introduction). Reintroduce the flies to the T-maze as in stage 4.7.</li>
	<li>For long-term memory, use a custom built maze which allows several batches of flies to be trained simultaneously. Administer 5 cycles of training with a 15&#160;min&#160;inter-cycle interval (spaced) or without an inter-cycle interval (massed). Maintain the flies at 18 &#176;C and 70% humidity in the dark until testing. Prior to testing, move the flies to 25 &#176;C and allow them to acclimatize for at least 1 hr. Assess long-term memory 24 hr after training.</li>
	<li>After the behavioral experiments, clean the odor cups with hot water and odorless detergent. After drying, coat the cups with 10 &#956;l&#160;sigmacote. Dry the sigmacote by heating in a microwave oven. Occasionally clean the T-maze perspex tubes and odor blocks with hot water and odorless detergent.</li>
</ol>
5. Calculation of the Performance Index: a Measure of the Flies&#8217; Memory
<ol>
	<li>Calculate the Performance Index (PI) for each condition as the number of flies avoiding the shock-paired odor (CS-) minus the number of flies choosing the shock-paired odor (CS+) divided by the total number of flies (CS- + CS+)1. 
	Performance index (PI) = (# CS- flies - # CS+ flies) / (# total flies)</li>
	<li>Calculate the final PI of the experiment by averaging the PI of the experiment in which MCH was the shock-paired odor and one in which OCT was the shock-paired odor. This removes any bias of the flies having a higher preference for one odor.</li>
</ol>
6. Sensorimotor Controls
<ol>
	<li>Perform odor acuity by introducing ~40-50 flies into the T-maze6,7,17.</li>
	<li>After 90 sec, move the flies to the choice point, and allow them 2 min&#160;to choose between pure odors and air.</li>
	<li>Collect and count the flies. Calculate the avoidance percentage by dividing the total number of flies that chose the odor by those that participated in the test.</li>
	<li>For shock reactivity6,7,17, introduce the flies into the shock chamber.</li>
	<li>After 90 sec of rest, administer a 60-V DC electric shock, from which the flies can escape to a similar tube without a shock.</li>
	<li>Allow 2 min&#160;for the flies to choose; collect and count the flies. Calculate the shock avoidance percentage by dividing the number of flies that avoided the shock by escaping the shock tube by the total number of flies in the experiment. Include the flies that remain in the central chamber in the total of those that escaped the electric shock.</li>
</ol>

<ol>
	<li>Tully, T., Quinn, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+and+retention+in+normal+and+mutant+Drosophila+melanogaster.">Classical conditioning and retention in normal and mutant Drosophila melanogaster.</a> Journal of Comparative Physiology A. 157, 263-277 (1985).</li><li>Bolduc, F. V., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+and+intellectual+disability.">Fruit flies and intellectual disability.</a> Fly (Austin). 3, 91-104 (2009).</li><li>McGuire, S. E., Deshazer, M., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty+years+of+olfactory+learning+and+memory+research+in+Drosophila+melanogaster.">Thirty years of olfactory learning and memory research in Drosophila melanogaster.</a> Prog Neurobiol. 76, 328-347 (2005).</li><li>Keene, A. C., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+olfactory+memory:+single+genes+to+complex+neural+circuits.">Drosophila olfactory memory: single genes to complex neural circuits.</a> Nat Rev Neurosci. 8, 341-354 (2007).</li><li>Chiang, H. C., Wang, L., Xie, Z., Yau, A., Zhong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3+kinase+signaling+is+involved+in+Abeta-induced+memory+loss+in+Drosophila.">PI3 kinase signaling is involved in Abeta-induced memory loss in Drosophila.</a> Proc Natl Acad Sci USA. 107, 7060-7065 (2010).</li><li>Kanellopoulos, A. K., Semelidou, O., Kotini, A. G., Anezaki, M., Skoulakis, E. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+deficits+consequent+to+reduction+of+the+Fragile+X+mental+retardation+protein+result+from+metabotropic+glutamate-mediated+inhibition+of+cAMP+signalling+in+Drosophila.">Learning and memory deficits consequent to reduction of the Fragile X mental retardation protein result from metabotropic glutamate-mediated inhibition of cAMP signalling in Drosophila.</a> J Neurosci. 32, 13111-13124 (2012).</li><li>Malik, B. R., Gillespie, J. M., Hodge, J. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CASK+and+CaMKII+function+in+the+mushroom+body+a&#8217;/&#223;&#8217;+neurons+during+Drosophila+memory+formation.">CASK and CaMKII function in the mushroom body a&#8217;/&#223;&#8217; neurons during Drosophila memory formation.</a> Front Neural Circuits. 7, 52 (2013).</li><li>Gerber, B., Stocker, R. F. <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+larva+as+a+model+for+studying+chemosensation+and+chemosensory+learning:+a+review.">The Drosophila larva as a model for studying chemosensation and chemosensory learning: a review.</a> Chem Senses. 32, 65-89 (2007).</li><li>Gillespie, J. M., Hodge, J. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CASK+regulates+CaMKII+autophosphorylation+in+control+of+synaptic+growth+and+appetitive+learning.">CASK regulates CaMKII autophosphorylation in control of synaptic growth and appetitive learning.</a> Front Molecular Neuroscience. 6, 27 (2013).</li><li>Apostolopoulou, A. A., Widmann, A., Rohwedder, A., Pfitzenmaier, J. E., Thum, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetitive+associative+olfactory+learning+in+Drosophila+larvae.">Appetitive associative olfactory learning in Drosophila larvae.</a> J Vis Exp. (72), e4334 (2013).</li><li>Tonoki, A., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+impairs+intermediate-term+behavioral+memory+by+disrupting+the+dorsal+paired+medial+neuron+memory+trace.">Aging impairs intermediate-term behavioral memory by disrupting the dorsal paired medial neuron memory trace.</a> Proc Natl Acad Sci USA. 109, 6319-6324 (2012).</li><li>Yamazaki, D., Horiuchi, J., Nagano, S., Tamura, T., Saitoe, M. <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+DCO+mutation+suppresses+age-related+memory+impairment+without+affecting+lifespan.">The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan.</a> Nat Neurosci. 10, 478-484 (2007).</li><li>Cavaliere, S., Malik, B. R., Hodge, J. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ+channels+regulate+age-related+memory+impairment.">KCNQ channels regulate age-related memory impairment.</a> PLoS One. 8, e62445 (2013).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Isabel, G., Pascual, A., Preat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exclusive+consolidated+memory+phases+in+Drosophila.">Exclusive consolidated memory phases in Drosophila.</a> Science. 304, 1024-1027 (2004).</li><li>Perisse, E., Burke, C., Huetteroth, W., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking+revelations+and+saccharin+sweetness+in+the+study+of+Drosophila+olfactory+memory.">Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory.</a> Curr Biol. 23, R752-R763 (2013).</li><li>Tully, T., Preat, T., Bonyton, S. C., Del Vecchio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+consolidated+memory+in+Drosophila.">Genetic dissection of consolidated memory in Drosophila.</a> Cell. 79, 35-47 (1994).</li><li>Lyons, L. C., Roman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+short-term+memory+in+Drosophila.">Circadian modulation of short-term memory in Drosophila.</a> Learning and memory. 16, 19-27 (2009).</li><li>Le Glou, E., Seugnet, L., Shaw, P. J., Preat, T., Gouguel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+consolidated+memory+retrieval+following+sleep+deprivation+in+Drosophila.">Circadian modulation of consolidated memory retrieval following sleep deprivation in Drosophila.</a> Sleep. 35 (10), 1377-1384 (2012).</li><li>Placais, P. Y., Preat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+favour+survival+under+food+shortage,+the+brain+disables+costly+memory.">To favour survival under food shortage, the brain disables costly memory.</a> Science. 339, 440-442 (2012).</li><li>Hirano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fasting+launches+CRTC+to+faciltate+long-term+memory+formation+in+Drosophila.">Fasting launches CRTC to faciltate long-term memory formation in Drosophila.</a> Science. 339, 443-446 (2012).</li><li>Schroll, C., 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=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16, 1741-1747 (2006).</li><li>Claridge-Chang, 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=Writing+memories+with+light-addressable+reinforcement+circuitry.">Writing memories with light-addressable reinforcement circuitry.</a> Cell. 139, 405-415 (2009).</li><li>Aso, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dopamine+pathways+induce+aversive+odor+memories+with+different+stability.">Three dopamine pathways induce aversive odor memories with different stability.</a> PLoS Genetics. 8, e1002768 (2012).</li><li>Connolly, J. B., Tully, T., Roberts, D. 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> Drosophila: a Practical Approach. , 265-319 (1998).</li><li>Connolly, J. 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=Associative+learning+disrupted+by+impaired+Gs+signaling+in+Drosophila+mushroom+bodies.">Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.</a> Science. 274, 2104-2107 (1996).</li><li>Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traces+of+Drosophila+memory.">Traces of Drosophila memory.</a> Neuron. 70, 8-19 (2011).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+kenyon+cells+in+situ.">Cholinergic synaptic transmission in adult Drosophila kenyon cells in situ.</a> J Neurosci. 26, 265-272 (2006).</li><li>Young, J. M., Wessnitzer, J., Armstrong, J. D., Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+and+non-elemental+olfactory+learning+in+Drosophila.">Elemental and non-elemental olfactory learning in Drosophila.</a> Neurobiol Learn Mem. 96, 339-353 (2011).</li><li>Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., Heberlein, U. .. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+model+for+alcohol+reward.">Drosophila model for alcohol reward.</a> Nat Neurosci. 14, 612-619 (2011).</li><li>Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+reveals+neural+circuit+mechanisms+of+fly+memory.">Dopamine reveals neural circuit mechanisms of fly memory.</a> Trends Neurosci. 33, 457-464 (2010).</li></ol>

The authors have nothing to disclose.

The Drosophila adult olfactory shock learning assay presented here allows analysis of the molecular mechanisms underlying different phases of memory, including long-term memory15-17. As well as determination of the effect of circadian rhythms18, sleep19, diet20,21, senescence11-13, neurodegenerative disease5 and drug treatments5,6,19 on memory.
Many powerful approaches have been recently developed for the functional imaging of the neural circuits that mediate olfactory memory in flies3,4,7,11,16,27. These optogenetic techniques use the vast repertoire of the different promoters available in Drosophila14,16. These promoters are used to express genetically encoded calcium and cAMP reporters in the memory neurons16,27 to study the effect of specific gene mutations on memory traces.
The use of conditional promoters and mutations in adults allows the study of the post-developmental role of a gene product in memory3,4,6,7,13,14. Imaging and behavioral approaches can be combined with light- and heat-activated channels to stimulate or inhibit different neurons in the memory circuit11,14,16,22-24 to further elucidate their function. Furthermore, mushroom body memory neurons are accessible to whole-cell patch clamp recordings28, and mathematical and computational techniques are being used to model Drosophila olfactory memory29.
These experimental advances, combined with the different forms of associative memory protocols introduced here, allow Drosophila to be used to model the molecular- and circuit-level changes in associative memory that occur in response to reward, punishment, motivation, addiction, aging and disease5,6,11-13,16,30-31.

The method presented here is that described by Tully and Quinn with some small modifications1. The experiment is performed in two phases: the flies are trained in the first phase, and the trained flies are tested in the second phase. During training, a group of flies are simultaneously exposed to odor 1 (CS+) and an electric shock (US) in a training tube. The flies then receive odor 2 (CS-) without an electric shock. This single pairing of a particular odor with a shock is called 1-cycle training, and the odors that are most frequently used are 4-methylcyclohexanol (MCH) and 3-octanol (OCT).
One-cycle training leads to the formation of a labile phase of memory that can be detected for up to 7 hr; however, memory is typically tested immediately to determine what is termed learning, acquisition or 2&#160;min&#160;memory. Memory measured at 30 min&#160;or 1 hr is referred to as short-term memory, whereas 3&#160;hr memory is referred to as mid-term memory. The exposure of flies to repetitive training cycles with gaps between the training cycles (spaced training) leads to a consolidated form of long-term memory that is CREB transcription dependent and lasts up to a week. Training without gaps (massed training) leads to the formation of anesthesia-resistant memory (ARM), which similar to long-term memory, is typically measured 24 hr after 5 cycles of training7,13,15-17,20,21.
With this approach, the effect of various gene mutations on these different phases of memory can be determined. The promoter-driven expression of light- or temperature-sensitive transgenes to activate or block the neural activity of specific neurons allows one to investigate which neurons are required for memory acquisition, consolidation and retrieval3,4,11,15,16,20,22-24. Memory at 1 hr is typically measured when studying age-related memory impairment because this form of memory appears particularly vulnerable to the effects of ageing11-13. A full range of behavioral and genetic controls are performed with memory experiments, for example, to determine whether a performance defect is because of a central memory defect or a peripheral sensory defect that prevents the fly from sensing the shock or olfactory cue5-7,17,25,26.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:189px;height:28px;">Materials</td>
			<td style="width:139px;height:28px;">Source</td>
			<td style="width:116px;height:28px;">Cat. No.</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">3-Octanol</td>
			<td style="width:139px;height:20px;">Sigma</td>
			<td style="width:116px;height:20px;">218405</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">4-Methyl cyclohexanol</td>
			<td style="width:139px;height:20px;">Sigma</td>
			<td style="width:116px;height:20px;">15,309-5</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">Benzaldehyde</td>
			<td style="width:139px;height:20px;">Sigma</td>
			<td style="width:116px;height:20px;">418099</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">Mineral Oil</td>
			<td style="width:139px;height:20px;">Fluka</td>
			<td style="width:116px;height:20px;">BP2629-1</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">Hexyl acetate</td>
			<td style="width:139px;height:20px;">Sigma</td>
			<td style="width:116px;height:20px;">108154</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:20px;">Fructose</td>
			<td style="width:139px;height:20px;">Sigma</td>
			<td style="width:116px;height:20px;">F0127</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:189px;height:22px;">Agarose</td>
			<td style="width:139px;height:22px;">Bioline</td>
			<td style="width:116px;height:22px;">BIO-41025</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The performance index (PI) serves as a measure of memory. Table 1 illustrates a representative calculation of PI.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCH paired with shock</td>
			<td>3-OCT paired with shock</td>
		</tr>
		<tr>
			<td>Flies avoiding MCH (in OCT tube) = 80 
			Flies preferring MCH (in MCH tube) = 20 
			PI1- (80-20)/80+20) 
			= 0.6</td>
			<td>Flies avoiding OCT (in MCH tube) = 75 
			Flies preferring OCT (in OCT tube) = 25 
			PI2&#160;= (75-25)/(75+25) 
			=0.5</td>
		</tr>
		<tr>
			<td>PI of the experiment= (0.6+0.5)/2=0.55</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1.&#160;A representative calculation of performance index using illustrative data.&#160;Performance indices for different experiments can be compared to elucidate memory effects. Once such comparison is shown in Figure 3, which contains the results from a series of experiments performed with Canton S wildtype adult flies (WT) and dunce learning mutant adult flies1. The mean of 10 PIs is provided, with error bars representing the standard error of mean (SEM). These results demonstrate that dunce flies show a reduction in learning compared to wildtype.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50107/50107fig1highres.jpg" src="/files/ftp_upload/50107/50107fig1.jpg" /> 
Figure 1. The adult experimental set-up. The flies are trained and tested in a T maze. The training involves presenting an odor A with electric shock followed by a second odor B without electric shock. After a rest period in the middle chamber the flies are presented with both the odors simultaneously. The flies are trapped in the two tubes and collected and counted to obtain learning/memory scores.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50107/50107fig2highres.jpg" src="/files/ftp_upload/50107/50107fig2.jpg" /> 
Figure 2. The adult training protocol. The flies are training in two steps. The first step where flies receive an odor (CS+) paired with electric shock (US) for 60 sec. In the next step flies receive a second odor (CS-) without electric shock. Flies are then allowed to rest for 90 sec after which they are tested for their choice between CS+ and CS-.
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/50107/50107fig3highres.jpg" width="400" /> 
Figure 3. A representative graph showing dunce and wildtype learning in adult Drosophila. WT and dunce flies were tested following one session of training. dunce flies show a reduction in learning compared to the WT (n = 10).

Watch this Scientific Journal Video about Drosophila Adult Olfactory Shock Learning at JoVE.com

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 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 ...

drosophila-adult-olfactory-shock-learning

Research

JoVE Journal

Neuroscience

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 ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

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.

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.

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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 ...

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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

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.