1. Behavioral Analysis in
Drosophila melanogaster
Lawrence T. Reiter, Ph.D.
Associate Professor
Department of Neurology
lreiter@uthsc.edu
901-448-2635
2. Why study fruit flies?
Short Generation Time and Lifespan
Thousands of Flies Can Be Analyzed at Once (Good Statistics)
Powerful Molecular and Genetic Tools Available
Simple Nervous System for Analysis (Single Neuron Knock Outs)
100 Years of Fly Genetics!
3. Comparison of
Model Genetic Organisms
Organism Transcriptome
(Genes)
*Similarity to
Human Genome
Cellular
Complexity
Organisms per
Genetic Screen
Generation
Time
Yeast 6,200 46% 1 cell >109 2 hours
Nematode 18,300 43% 959 cells 106-107 3 days
Fly 14,400 61% >106 cells 105-106 10 days
Mouse >30,000 >95% >109 cells 102-103 12 weeks
* E-value ≤ 0.001, IHGSC, Nature 409, p903.
4. Do Fruit Flies Have Behaviors?
Courtship behaviors (memory and learning; communication)
Visual behaviors (phototaxsis and visual acuity)
Olfaction and Taste (memory and learning)
Geotaxis (ageing as well as adaptation to environment)
Locomotion (Dopamine assays)
Alcohol and Cocaine resistance/sensitivity (addiction behaviors)
Grooming (restricted/repetitive behavior)
Social Interaction (autism social space defects)
5. GAL4-UAS Expression
System
Genomic Tissue
Specific Enhancer
(e.g. eye Specific)
Cross Responder and
Driver Stocks
Selectable
Marker
GAL4
Brain Eye Wing Heatshock
GAL4 Drivers Active in Different Tissues
Result:
Expression of cDNA in eye
Inverted UAS
Repeat
Inverted
Repeat
Selectable
Marker
cDNA
Fly Lines Carrying UAS-psn,
UAS-UBE3A, UAS-APP, etc.
6. dBrainbow
Hampel, S. et al. Drosophila Brainbow: a recombinase-based fluorescence labeling
technique to subdivide neural expression patterns. Nat. Methods 8, 253–259 (2011).
8. orb2 is essential for long-term
courtship memory
CITATION: Keleman et al. Nature Neuroscience. 12: 1587-93.
PROBLEM: Which neurons are involved in memory and learning? Are there long
term and short term circuits in the fly brain?
BEHAVIOR: Courtship memory. Males remember when they are rejected by
females which have already mated.
ASSAY:
1. Courtship index: number of times a male courts in 10 min.
2. Learning index: the percentage reduction mean courtship behavior.
CONCLUSIONS:
1. orb2 mutants are defective for long term, but not short term courtship
memory.
2. Long term memory can be rescued in orb2 mutants through expression of
UAS-orb2 in gamma neurons of the mushroom body.
9.
10.
11. Age-dependent climbing speed
CITATION: Rhodenizer et. al. 2008. Experimental Gernontology. 43: 739-48.
PROBLEM: Reduction in walking speed in humans is a clear risk factor for early
death in older individuals.
BEHAVIOR: Negative Geotaxis – climbing up the vial.
ASSAY: RING – Rapid Iterative Negative Geotaxis
CONCLUSIONS:
1. Negative geotaxis is a climbing (not jumping behavior).
2. Climbing speed is constant at a given age and genetic background.
3. Climbing speed decreases with age and response latency increases with age.
14. Painless is involved in nociception in
adult flies.
CITATION: Xu et al. Genes, Brain and Behavior. 5: 602-613.
PROBLEM: Can you assay nociception (pain perception) in flies?
BEHAVIOR: Various tests of noxious heat using a CO2 laser and hotplate. Also
tested jump latency.
ASSAY:
1. CLB (CO2 laser heat applied to abdomen of flies).
2. HP (fly is placed on a hotplate).
3. Jump Test (fan is used to elicit jump response in flies.
CONCLUSIONS:
1. painless mutants are defective for nociception.
2. painless is expressed in neurons of the femurs, tarsal segments and
mushroom body.
3. The mushroom body is NOT needed for nociception.
18. Flies provide a method to investigate the
genetics of ethanol and cocaine addiction.
CITATION: Rothenfluh and Heberlein. 2002. Current Opinion in Neurobiology. 12:
639-45.
PROBLEM: Can we use the genetic screening power of Drosophila to identify
genes involved in alcohol and cocaine addiction?
BEHAVIOR: Activity and movement assays. Have also used cones to screen.
ASSAY:
1. Expose flies to drug vapors and record activity.
2. Expose flies to alcohol vapor and let them settle in a set of cones
(inebriometer).
CONCLUSIONS:
1. Several genes have been identified for ethanol sensitivity and tolerance:
rutabaga, amnesiac, cheap date, fasciclin II…
2. Periodicity genes like clock, timeless and cycle are required for cocaine
sensitivity.
22. Dube3a regulates dopamine through
transcriptional regulation of Punch.
CITATION: Ferdousy et al. 2011. Neurobiology of Disease. 41(3): 669-77.
PROBLEM: Punch protein is up-regulated when Dube3a is over-expressed in fly
heads. Can increased or decreased Dube3a have a direct effect on dopamine
induced behaviors in flies?
BEHAVIOR: Activity assays in flies expressing wild type, mutant and RNAi
constructs for Dube3a.
ASSAY:
1. Heatshock-Gal4 and C155-Gal4 used to express the following:
• UAS-Dube3a
• UAS-Dube3a-C/A (can txpt activate, but not ubiquitinate)
• UAS-Dube3a-RNAi
CONCLUSIONS:
1. Dube3a can regulate the products of GTP Cyclohydrolase I (Punch)
2. Dube3a can regulate dopamine levels.
3. This regulation is NOT related to the ubiquitination of Punch (may be
transcriptional regulation).
24. Punch Products
Each sample contained 80–150 flies with a male: female ratio of 1:1. All flies were 3-5 days old. Statistics
are one-way Anova with a Bonferroni post test for statistical analysis (*, p<0.05, **, p<0.01, ***, p<0.001).
25. Dopamine Levels
Each sample contained 80–150 flies with a male: female ratio of 1:1. All flies were 3-5 days old. Statistics
are one-way Anova with a Bonferroni post test for statistical analysis (*, p<0.05, **, p<0.01, ***, p<0.001).
26. Males
Females
Activity Assays
All flies were 3-5 days old (n=15-20). Statistics are one-way Anova with a Bonferroni post test for statistical
analysis (*, p<0.05, **, p<0.01, ***, p<0.001).
27. Automated quantification of locomotion, social interaction,
and mate preference in Drosophila mutants
CITATION: Iyengar et al. J. Neurogenetics. 26: 306-316.
PROBLEM: How can we measure multiple flies at once for locomotion and social
interactions?
BEHAVIOR: Auto tracking system used in flies. Analysis of motor activity in the K+
and Na+ channel mutants, Hk1 and parats1
ASSAY:
1. Measure activity
2. Measure social interaction over time in a natural environment.
CONCLUSIONS:
1. Hk mutants are more active and para mutants less active.
2. IowaFLI Tracker can be employed to explore motor coordination and social
interaction phenomena in behavioral mutants of Drosophila.
30. fADOS: Autism Behavior Assessment in
Flies.
CITATION: Grant in submitted to NIH NINDS.
PROBLEM: How can we assess autistic behaviors in flies that represent the three
descriptive domains of autism: defects in communication, repetitive/restrictive
behaviors and social interaction defects?
BEHAVIORS: 1) Mating Assay; 2) Grooming Assay; 3) Social Space Assay
ASSAYS:
1. Mating:
a) Ability for mutant males to mate with females (Expressive Comm)
b) Ability for mutant females to mate with males (Receptive Comm)
2. Grooming
a) Amount of time spent grooming each body part (OCD-like)
b) Total amount of time spent grooming (Restrictive Repetitive Behavior)
3. Social Space Assay
a) Using the SSI and the social space triangle – see paper.
31. Mating Behaviors
FROM: Krstic D, Boll W, Noll M (2009) Sensory Integration Regulating Male
Courtship Behavior in Drosophila. PLoS ONE 4(2): e4457.
32. Repetitive Grooming Behaviors
Sample video of dfmr1 mutant grooming activity. A 15 day-old dfmr1 mutant fly initially
explores the environment for 10 s, but then begins grooming excessively.
FROM: Tauber JM, Vanlandingham PA, Zhang B (2011) Elevated Levels of the Vesicular
Monoamine Transporter and a Novel Repetitive Behavior in the Drosophila Model of Fragile
X Syndrome. PLoS ONE 6(11): e27100.
33. Fragile X Mutants Groom More
Tauber JM, Vanlandingham PA, Zhang B (2011) Elevated Levels of the Vesicular Monoamine Transporter and a
Novel Repetitive Behavior in the Drosophila Model of Fragile X Syndrome. PLoS ONE 6(11): e27100.
34. Social Space Index (SSI)
Simon et al (2012). Genes, Brain and Behavior Volume 11, Issue 2, pages 243-252, 23
35. Assignment – Thursday in class
• Perform Social Space Assay on flies from the
DGRP collection
• Analyze SSI using your cell phone and ImageJ
36. Additional Information
Lords of the Fly: Drosophila Genetics and the Experimental Life (Paperback)
by Robert E. Kohler
Genetics of Alcohol-Induced Behaviors in Drosophila.: An article from: Alcohol
Research & Health
by Ulrike Heberlein
Time, Love, Memory: A Great Biologist and His Quest for the Origins of Behavior by
Jonathan Weiner
Fly Pushing: The Theory and Practice of Drosophila Genetics by Ralph J. Greenspan
Citations:
1) Rhodenizer et. al. 2008. Experimental Gernontology. 43: 739-48.
2) Keleman et al.2007. Nature Neuroscience. 12: 1587-93.
3) Xu et al.2006. Genes, Brain and Behavior. 5: 602-613.
4) Ferdousy et al. 2011, Neurobiology of Disease. 41(3): 669-77.
5) Rothenfluh and Heberlein. 2002. Current Opinion in Neurobiology. 12: 639-45.
6) Krstic et al. 2009 PLoS ONE 4(2): e4457.
7) Tauber et al. 2011 PLoS ONE 6(11): e27100.
