Why am I working in a Drosophila lab? Dr. Stefan Pulver, Brandeis University
!Short life span The advantages of Drosophila as a model organism in neurocience!genetic manipulations are relatively easy!forward genetic screens for behavior mutants are possible with a minimum of effort.!techniques for testing a wide range of behaviors are highly refined and possible with limited space and resources.!virtually all of the same neurotransmitters, ion channels, synaptic proteins, signaling molecules, and receptors as vertebrate nervous systems
Genetic screens for behavior mutants Expose flies to mutagens Many flies die Some flies live Screen survivors for defects in specific behaviors An unbiased approach! Make mutant lines Figure out what gene and/or genes are messed up
What popped out?!100 s of mutant lines with defects in specific behaviors!many lines with defects mapped to a single gene locus (!) shaker (locomotor) slowpoke (locomotor) period (circadian) fruitless (courtship)!massive # of genes important for nervous system function identified, cloned!hard evidence for the idea that genes play an important role in determining animal behavior (.Does this hold true for humans?)
What are the disadvantages of Drosophila as a model system?
Circuit analysis in Drosophila lags behind other model organisms Genetics Land in Fly World (Brightly lit, suburbanized) Behavior Land in Fly World (Brightly lit, suburbanized) Circuit Land in Fly World (Dark, pixelated) Why is this?
Fly brains are small Fly brain Fly head Crab ganglia Fly CNS
Why bother starting over in Drosophila? Promotor! GAL4! X! UAS-GFP! Promotor! GAL4! GFP! GAL4! UAS-GFP! Spatial control of gene expression!
Combine GAL4-UAS with excitability manipulations Bacterial sodium channel Nitabach et al., 2006 What s going to happen when we put this in a neuron?
Something unexpected! Sheeba et al., 2008
Genetic inhibition of Na/K pump function Sun et al., 2001 Lingrel and Kuntzweiler, 1994 ***Changed G to A at phosphorylation site required for ATP hydrolysis What s going to happen when we inhibit Na/K pump function in a neuron?
How does inhibiting Na + /K + pump affect excitability? -50 mv control Inhibited Na/K pump in larval motor neuron (UAS-dnATPase)
How are we going to use this new tool? Inhibiting Na/K pump function can be used to hyper-excite Fly sleep circuit cells PDF-GAL4 alone (N=34) UAS-dnATPase alone (N=33) PDF-GAL4 + UAS-dnATPase (N=28) LP DP Parisky et al. (2008) Neuron
Limitations to this approach? Chronic activation throughout development! What other tools are available?
Two new tools for acutely activating neural circuits CHANNELRHODOPSIN-2 ion channels!light activated cation channel!evokes action potentials when activated in neurons!can be targeted to subsets of cells with GAL4 / UAS dtrpa1 ion channels!warmth and voltage gated cation channel!large inward currents in response to warming!endogenous expression in only a few cells!can be targeted to subsets of cells with GAL4 / UAS
TEMPTATION: Immediately use tools in behavior experiments RESIST TEMPTATION: First, make effort to understand how tools work
Mutated ChR2 makes cells more responsive to blue light Point mutation in pore region! Whole cell patch
How do stimulation dynamics affect firing rate? Whole cell patch
How does sensory neuron activation affect crawling? GAL4 alone UAS alone GAL4 + UAS (old ChR2) GAL4 alone UAS alone GAL4 + UAS (mutated ChR2) Stan Pashkovski
How do dtrpa1 expressing cells respond to warming? Hamada et al. (2008), Nature
GAL4-UAS controls look good
How does dtrpa1 adaptation compare to ChR2 adaptation? NMJ prep
What s an important control to do? Does dtrpa1 expression cause any changes @ 22 o C? Whole cell patch
dtrpa1 activation of motor and sensory circuits inhibits crawling dtrpa1 in larval sensory neurons Stan Pashkovski
How does dtrpa1 activation in PDF neurons affect sleep behavior? Parisky et al. (2008), Neuron
Welcome To The Maggot Intact Larva-> Dissected Larva-> Recording
The Larval Neuromuscular Junction (NMJ)!Action Potential depolarizes presynaptic terminal!calcium influx causes release of vesicles containing glutamate into synaptic cleft!neurotransmitter release causes postsynaptic stimulation and Excitatory Junction Potential (EJP) Neuroscience, Purves et. al
Why work on the Larval NMJ?! Similar to excitatory synapses in mammalian CNS.! Both Drosophila and Mammalian synapses utilize glutamate and display similar patterns of development! Many of the molecules have their functions conserved across systems.
!!! Channel Rhodopsin-2 New and Improved! Traditionally, we use suction-electrode stimulation to evoke EJP Combination of ChR2 and genetic expression techniques, we can remotely activate spikes in subsets of motor neurons Physiology rigs retrofitted for controlling LED stimulation
Goal: Quantal analysis! Used to determine the number of vesicles released per action potential! Must first measure the post-synaptic depolarization caused by a single vesicle (Quantum).
Quantal Analysis!!! Determine the size of the quanta (minis) Determine the size of EJPs and determine quantal content of light evoked EJP Analyze quanta per EJP based on EJP amplitude
Brand New!! First time this experiment has been run in a teaching lab this size! Please give us your honest thoughts! Please be gentle with LEDs