reiter lecture 11.11.14

Behavioral Analysis inDrosophila melanogaster

Lawrence T. Reiter, Ph.D.Associate Professor

Department of Neurologylreiter@uthsc.edu

901-448-2635

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!

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.

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)

GAL4-UAS Expression

System

Cross Responder and

Driver Stocks

Genomic Tissue

Specific Enhancer

Selectable

Marker

GAL4

Brain Eye Wing Heatshock

GAL4 Drivers Active in Different Tissues

(e.g. eye Specific)

Result:

Expression of cDNA in eye

UASInverted

Repeat

Inverted

Repeat

Selectable

Marker

cDNA

Fly Lines Carrying UAS-psn,

UAS-UBE3A, UAS-APP, etc.

dBrainbow

Hampel, S. et al. Drosophila Brainbow: a recombinase-based fluorescence labeling

technique to subdivide neural expression patterns. Nat. Methods 8, 253–259 (2011).

Janelia Farms FlyLight Project

Cell Reports 2012 2, 991-1001DOI: (10.1016/j.celrep.2012.09.011)

Copyright © 2012 The Authors Terms and Conditions>6000 GAL4 Lines

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.

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.

Genetic Background

1-7 Weeks

Height

Speed

Latency

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.

CLB

HP

JT

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.

ETHANOL

COCAINE

http://anatomy.ucsf.edu/heberlein/Research.htm

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).

Dopamine Pathway

GTP H2NeopterinP3

GTPCH1 (Punch)

BH4 Tyrosine

Tyrosine L-Dopa

TH (pale) Ddc

Dopamine

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).

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).

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).

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 activity2. 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.

The IowaFLI Tracker System

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. Groominga) Amount of time spent grooming each body part (OCD-like)b) Total amount of time spent grooming (Restrictive Repetitive Behavior)

3. Social Space Assaya) Using the SSI and the social space triangle – see paper.

Mating Behaviors

FROM: Krstic D, Boll W, Noll M (2009) Sensory Integration Regulating Male Courtship Behavior in Drosophila. PLoS ONE 4(2): e4457.

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.

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.

Social Space Index (SSI)

Simon et al (2012). Genes, Brain and Behavior Volume 11, Issue 2, pages 243-252, 23

Assignment – Thursday in class

• Perform Social Space Assay on flies from the DGRP collection

• Analyze SSI using your cell phone and ImageJ

Additional InformationLords 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 & Healthby 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.