Optogenetics. how to use light to manipulate neuronal networks. Alexandra Götz

Transcription

1 Optogenetics how to use light to manipulate neuronal networks Alexandra Götz 1

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3 Outline Background Neuron and ion channels Action potential Light sensitive channels Channel induction ReaChr Experiment Results ReaChR in Drosophila CNS neurons Wing extension reflex Changes due to social factors Summary Future challenges 3

4 Neuron and ion channels 4

5 Action potential (9) 5

6 Light sensitive channels (3) 6

7 Light sensitive channels (3) 7

8 How are the light sensitive channels induced in the brain? 8

9 (8) 9

10 ReaChR Red-activable Channelrhodopsin Improved signaling characteristics e.g. greater photocurrent and faster kinetics λ~ nm (can penetrate deeper into tissue) Experiments without tissue damage possible Drosophila as testsubject? 10

11 Experiment Study freely moving test subjects Led light with different: wavelength cw vs pulsed ReaChR channels in different neuron types (1) 11

12 ReaChR in Drosophila Gr5a receptor neurons studying PER (proboscis extension reflex) comparison of different channel types ReaChR channel: Only light sensitive channel leading to robust response to green (530nm) and red (627nm) light dtrpa1 channel (thermal activated): No response at high surrounding T 12

13 ReaChR in Drosophila continuous illumination: PER decays exponentially depolarization block in Gr5a neurons (cf. thermal channels) pulsed illumination (1Hz): repeated spiking when light is activated (2) 13

14 CNS neuron activation: Testing different neuron types in CNS (central nervous system) with red and blue light-sensitive channels Various behavioral responses expected in channels e.g: wing extension reflex (male courtship song) response for ReaChR channel (green and red) no response for blue channel (CNS too deep in tissue) LED tuned behavior; experiments tunable with: light intensity pulse frequency 14

15 Wing extension reflex 2 types of neurons known to trigger wing extension reflex, P1 and pip10, but their role is not nown yet: (1) (2) 15

16 Wing extension reflex 2 types of neurons known to trigger wing extension reflex, P1 and pip10, but their role is not nown yet: (1) (2) 16

17 Wing extension reflex neuron type P1 neuron pip10 control type probabilistic deterministic behavior response during illumination period 17,5 ± 27,5sec triggered with LED start 0,08 ± 0,04sec But both neuron types with ReaChR led to intensity independant behavior response. 17

18 Changes due to social factors: Probabilistic behavior of neurons leads to assumption that external factors could lead to changes in response. Test: single-housing males for 7 days Results: lower LED intensity leads to wing extension higher responses to stimulation Induction of ReaChR in both neurons separately indicates: P1 neurons are responsible for encanced sensitivity 18

19 Summary ReaChR is fitting opsin for Drosophila studies high-throughput screening of behavior possible reduced visual artifacts due to red illumination intensity and frequency dependent behavior probabilistic (P1) vs. deterministic (pip10) response social isolation can affect optogenetic behavior 19

20 Future challenges: finding other social impact factors to P1 or other neuron types ReaChR and other opsin engineering for faster kinetis and smaller activation light ranges create red-shifted inhibitory opsins study of interacting neurons activated with different wavelengths 20

21 Thanks for listening. Questions? 21

22 References: (1)Inagaki et al., Nat Methods March; 'Optogenetic control of freely behaving adult Drosophila using a red-shifted channelrhodopsin' (2) ; Supplementary info; ( ) (3)Fenno et al., Annual Rev. Neuroscience 2011; 'The Developement and Application of Optogenetics' (4)Lin et al.; Nat Neurosci October; 'ReaChR: A red-shifted variant of channelrhodopsin enables deep transcriptional optogentic excitation' (5)Yizhar et al.; Neuron 2011 July; 'Optogenetics in Neural Systems' (6) (7) (8) (9) (10) 22