Use of optics & Use of genetics Optogenetics Use of optics & Use of genetics https://www.tcd.ie/comparativemedicine/assets/pdf/events/charles_river-optogenetics_may15.pdf http://www.livescience.com/29340-optogenetics-brain-research-breakthrough-nsf-bts.html

Transgenic approaches are easy with fruit flies http://www.domyownpestcontrol.com/images/content/fruit_fly_up_close_1.JPG www.domyownpestcontrol.com

-Transgenes are targeted to specific tissues with GAL-4xUAS system. 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.

-Transgenes are targeted to specific tissues with GAL-4xUAS system. 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.

https://www.tcd.ie/comparativemedicine/assets/pdf/events/charles_river-optogenetics_may15.pdf http://es.slideshare.net/marinagoc/optogentica

http://jn.physiology.org/content/101/6/3075

http://www.nature.com/nmeth/journal/v5/n10/full/nmeth.1252.html

-Transgenes are targeted to specific tissues with GAL-4xUAS system. 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 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? Drosophila vesicular glutamate transporter gene (DVGLUT) that is expressed almost exclusively in neurons that release glutamate (Mahr and Aberle 2006).

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

What possibilities could this have for humans ? 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 ?

Felicitas Koch, Cooper Lab 2015 Mechanism of Channelrhodopsin2 in motor neurons Channelrhodopsin2 inactivated (normal light) Na+ Ca2+ ChR2 Felicitas Koch, Cooper Lab 2015 NO Muscle contraction

Mechanism of Channelrhodopsin2 in motor neurons Channelrhodopsin2 activated (blue light) Blue light Na+ Ca2+ ChR2 Glutamate Glutamate instead of Acetylcholine in Drosophila Muscle contraction Felicitas Koch, Cooper Lab 2015

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 Here show a movie

Body wall contractions in lavrae Dim or regular light /1min   Low intensity blue light /1min High intensity blue 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 Show a movie

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 Show a movie

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 Perform the assay before light exposure. Expose the fly to blue light for 10 sec Perform the assay 10 times. Between each trial there should be ONE minute rest.