Why am I working in a Drosophila lab? Dr. Stefan Pulver, Brandeis - - PowerPoint PPT Presentation

Why am I working in a Drosophila lab?

• Dr. Stefan Pulver, Brandeis University

The advantages of Drosophila as a model

• rganism in neurocience
• Short life span
• Genetic manipulations are relatively easy
• Forward genetic screens for behavior mutants are possible with a minimum
• f 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 Make mutant lines Figure out what gene and/or genes are messed up

An unbiased approach!

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

Behavior Land in Fly World (Brightly lit, suburbanized) Genetics Land in Fly World (Brightly lit, suburbanized) Circuit Land in Fly World (Dark, pixelated) Why is this?

Fly brains are small Crab ganglia Fly head Fly CNS Fly brain

Why bother starting over in Drosophila?

X

UAS-GFP GAL4

GAL4

UAS-GFP GFP

Promotor

GAL4

Promotor

Spatial control of gene expression!

Combine GAL4-UAS with excitability manipulations

What’s going to happen when we put this in a neuron?

Nitabach et al., 2006

Bacterial sodium channel

Sheeba et al., 2008

Something unexpected!

Genetic inhibition of Na/K pump function

Lingrel and Kuntzweiler, 1994 Sun et al., 2001 ***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?

Inhibited Na/K pump in larval motor neuron (UAS-dnATPase) control

• 50 mV

Parisky et al. (2008) Neuron

Inhibiting Na/K pump function can be used to hyper-excite Fly sleep circuit cells

LP DP

PDF-GAL4 alone (N=34) UAS-dnATPase alone (N=33) PDF-GAL4 + UAS-dnATPase (N=28)

How are we going to use this new tool?

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

Whole cell patch

Point mutation in pore region!

How do stimulation dynamics affect firing rate?

Whole cell patch

How does sensory neuron activation affect crawling?

Stan Pashkovski

GAL4 alone UAS alone GAL4 + UAS (old ChR2) GAL4 alone UAS alone GAL4 + UAS (mutated ChR2)

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

Does dTRPA1 expression cause any changes @ 22oC?

Whole cell patch

What’s an important control to do?

dTRPA1 activation of motor and sensory circuits inhibits crawling

Stan Pashkovski

dTRPA1 in larval sensory neurons

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