Transcript power point only here

Optogenetics
Use of optics
&
Use of genetics
http://www.livescience.com/29340-optogenetics-brain-research-breakthrough-nsf-bts.html
Transgenic approaches
are easy with fruit flies
www.domyownpestcontrol.com
Key Points
-Transgenes are targeted to specific tissues with
GAL-4xUAS system.
-Transgenes can be used to control or measure neural
activity.
-Active electrical properties of muscles are based on ion
channels.
Key Points
-Transgenes are targeted to specific tissues with GAL4xUAS system.
-Transgenes can be used to control or measure
neural activity.
-Active electrical properties of muscles are based on ion
channels.
Key Points
-Transgenes are targeted to specific tissues with GAL4xUAS system.
-Transgenes can be used to control or measure neural
activity.
-Active electrical properties of cells are based on
ion channels.
How could we target ChR to motor neurons?
OK371-Gal4 x UAS-ChR2
In other words, what gene is OK371?
Human Brain
https://en.wikipedia.org/wiki/Serotonin
Adult Drosophila brain with neurons stained green and the neurotransmitter serotonin stained red.
Green are serotonin containing neurons
http://www.mdpi.com/1422-0067/10/2/407/htm
Experiments
•
•
•
•
•
Larva & Adults
Neurons: Serotonin, dopamine, GABA
Motor neurons or sensory neurons
Examine acute behavioral changes
Think about the neural circuits and
influence on behavior
Summary
- Transgenes can be targeted to specific subsets of cells using binary
transcription activation
- Transgenes can be used to control neural activity and influence behaviors
-
What possibilities could this have for humans ?
-
What various types of experimental paradigms can be used to address
new questions ?
Mechanism of Channelrhodopsin2 in motor neurons
Channelrhodopsin2 inactivated (normal light)
Na+
Ca2+
ChR
2
Felicitas Koch, Cooper Lab 2015
NO Muscle contraction
17
Mechanism of Channelrhodopsin2 in motor neurons
Channelrhodopsin2 activated (blue light)
Blue light
Na+
Ca2+
ChR
2
Glutamate
Felicitas Koch, Cooper Lab 2015
Glutamate
instead
of
Acetylcholin
18
Muscle contractione
in
Blue light source setup
Group-1
UAS-ChR2-XXL (very sensitive ChR2 variant)
X D42-Gal4 (motor neuron driver)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Bodywall contractions in
larva
Body wall contractions in lavrae
Dim or regular
light /1min
Low intensity blue
light /1min
Dim or regular
light /1min
High intensity blue
light /1min
Dim or regular
light /1min
Group-1
• Negative geotaxic assay
• male and female cohorts plus and minus
ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8cm length, and the 14 cohort flies are transferred
to that empty marked vial. Another plastic vial is placed on top of the
marked one. The flies are left for one minute. The vials are tapped to knock
down the flies to the bottom of the tube. Then number of flies which climbed
across the 8 cm mark is recorded for 10 sec. This procedure is repeated
three times before light exposure and 6 times after light exposure (10 sec).
Group-1
Group-2
UAS-ChR2-XXL
X Gad1-Gal4 (GABAergic neurons)
- Body wall contractions in larvae
- Negative geotaxic assay in adults for both
minus and plus ATR and male and female
cohorts.
Group-2
UAS-ChR2-XXL
X Gad1-Gal4 (GABAergic neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Group-2
• Negative geotaxic assay
• male and female cohorts plus and minus ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8 cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(5 sec).
Group-3
UAS-ChR2-XXL
X ppk-GAl4 (type IV sensory neurons)
• Rolling behavior in larvae plus and minus ATR
• The rolling behavior is performed by placing a single
larva on the surface of an apple-juice agar plate. The
occurrence of rolling behavior is counted for 1st and 2nd
minute. The percentage of larvae that show rolling
behavior should be presented in a graphical form.
• Negative geotaxic assay foe adults male and femlae,
minus and plus ATR.
Group-3
• Negative geotaxic assay
• male and female cohorts plus and minus
ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(5sec).
Group-4
UAS-ChR2-XXL
X Trh-Gal4 (serotonergic neurons)
- Body wall contractions in larvae
- Negative geotaxic assay in adults for both
minus and plus ATR and male and female
cohorts.
Group-4
UAS-ChR2-XXL
X Trh-Gal4 (serotonergic neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Group-4
• Negative geotaxic assay
• male and female cohorts plus and minus
ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8 cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(15 sec).
Group-5
UAS-ChR2H134R-mcherry
X OK371-Gal4 (motor neurons)
- Body wall contractions in larvae
- Negative geotaxic assay in adults for both
minus and plus ATR and male and female
cohorts.
Group-5
UAS-ChR2H134R-mcherry
X OK371-Gal4 (motor neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Group-5
• Negative geotaxic assay
• male and female cohorts plus and minus
ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(15sec).
Group-6
UAS-ChR2H134R-mcherry
X Trh-Gal4 (serotonergic neurons)
- Body wall contractions in larvae
- Negative geotaxic assay in adults for both
minus and plus ATR and male and female
cohorts.
Group-6
UAS-ChR2H134R-mcherry
X Trh-Gal4 (serotonergic neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Group-6
• Negative geotaxic assay
• male and female cohorts plus and minus
ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(15 sec).
Group-7
UAS-ChR2-XXL
X ple-Gal4 (dopaminergic neurons)
plus ATR
- UAS-ChR2-XXL plus and minus ATR
200µM
- Body wall contractions in larvae
- Perform touch assay (head, abdomen, tail)
Group-7
UAS-ChR2-XXL
X ple-Gal4 (dopaminergic neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Touch assay
Head touch
Abdomen touch
Tail touch
Responses: No response, pause, tail flip, turn right or left, backward movement,
C-shaped bend, rolling behavior.
Touch assay
Group-8
- UAS-ChR2-XXL
X ple-Gal4 (dopaminergic neurons)
plus ATR
- UAS-ChR2-XXL plus and minus ATR
200µM
- Negative geotaxic assay in adults
Group-8
UAS-ChR2-XXL
X ple-Gal4 (dopaminergic neurons)
• Negative geotaxic assay
• male and female cohorts plus ATR
•
•
•
The adult flies aged are to be anesthetized with ice or CO2.
The males and females are to be sorted out and transferred into separate
vials in cohorts of 14 flies. The flies should be left to recover for 24h before
running the experiments.
A plastic vial is marked at 8 cm length, and the 8-10 cohort flies are
transferred to that empty marked vial. Another plastic vial is placed on top of
the marked one. The flies are left for one minute. The vials are tapped to
knock down the flies to the bottom of the tube. Then number of flies which
climbed across the 8 cm mark is recorded for 10 sec. This procedure is
repeated three times before light exposure and 6 times after light exposure
(10 sec).
Group-9
UAS-ChR2-XXL
X Trh-Gal4 (serotonergic neurons)
- Body wall contractions in larvae
- Minus and plus ATR
- Count the body wall contractions for 5
minutes while you shine the light on the
larvae.
- Perform touch assay (head, abdomen, tail)
Group-9
UAS-ChR2-XXL
X Trh-Gal4 (serotonergic neurons)
• - Larval locomotion behavior (-ATR n=5, +ATR n=5)
•
Placing a single larva on an apple-juice agar plate. The larva is left for one
minute to acclimate to the new environment.
•
The body wall contractions are being counted for one minute (BWCs/min)
while the larva is being exposed to regular light or dim light. Then the body
wall contractions are counted for one minute while larvae are being exposed
to blue light (470 nm wavelength) (a dispersed-soda-can device).
•
Also, body wall contractions are being counted while the larva was being
exposed to focused focal blue light (a focused light through a microscope
eyepiece with a mounted LED).
Touch assay
Head touch
Abdomen touch
Tail touch
Responses: No response, pause, tail flip, turn right or left, backward movement,
C-shaped bend, rolling behavior.
Touch assay
Group10
1. UAS-ChR2-XXL
X D42-Gal4 (motor neurons)
2. UAS-ChR2-XXl control
• plus and minus ATR
Phototaxic assay-
-
A device with a 25 cm long plastic tube and light source at one end in a dim-light
room is used to assess the phototaxic behavior of the adult flies.
The tube is narrow enough not to allow the adults to fly but only walk along the tube.
Also the standard small LED maglight fits snuggly in one end. The male or female
flies are anesthetized by ice for 25-30 sec. Individual flies are placed in each
apparatus.
The flies are left to recover for at least 10 min. Each apparatus with individual fly,
which is positioned horizontally or vertically, is tapped until the fly fall to the bottom of
the tube, which was closed by a rubber stopper. The time the fly crossed 10 cm line
and 20 cm line is recorded.
Phototaxic assay
1. Perform the assay before light exposure.
2. Expose the fly to blue light for 10 sec
3. Perform the assay 10 times. Between each
trial there should be ONE minute rest.