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Electrophysiology meets Optogenetics


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Electrophysiology meets Optogenetics combining Axon pCLAMP™ with Andor Mosaic®

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Electrophysiology meets Optogenetics

  1. 1. Electrophysiology meets Optogenetics combining Axon pCLAMP™ with Andor Mosaic® Mark Browne PhD, Director, Systems DivisionDecember 13, 2012
  2. 2. Optogenetics meets Electrophysiology Andor Mosaic ® with Axon pCLAMP™ Mark Browne PhD, Director, Systems DivisionDecember 13, 2012
  3. 3. 1979: Francis Crick noted that a key challenge to neuroscientists is toimprove ability to control single cells among a network/population of cells.1999: UCSD – Kuffler Lecture Series • Crick stressed combining techniques in molecular biology and neuroscience “Basically the argument is that to understand a complex biological system one must be able to interfere with it both precisely and delicately, probably at all levels, but especially at the cellular and molecular levels.” 3
  4. 4. Optogenetics Nature MethodsMethod of Year 2010Deisseroth, Boyden and others exploit opsins (bacterial photo-proteins) in mammalian systems Optogenetics comes of age 4
  5. 5. Opsins are essentially optically controlled ion pumps. This means they can beused to polarize and depolarize neurons – “fire” and “silence” – enablingcontrol of brain circuitry.Optogenetic control can be paired with geneticallyencoded fluorescent biosensors (GEBs). Targetsinclude ions, metabolites, enzyme activities,protein conformation, membrane voltage. © Riken InstituteJust as Francis Crick suggested we can manipulateat molecular (DNA) and cellular level precisely anddelicately (optical patterns) and monitor response– via optical, electrical, behavioural…In combination, Opsins and GEBs can be used to perturb and monitor manyaspects of neuronal and glial biology, to visualize synaptic transmission andactivity-dependent signalling in vitro and in vivo. 5
  6. 6. Is a neuron a linear integrator? Soma SS ~ -70 mV Soma SS S=ΣD? IF S > TAH (+40 mV) IF S > TAH THEN Fire (AP)1. Neurons are the basic building blocks of the nervous system. These specialized cells are the information-processing units of the brain responsible for receiving and transmitting information. Each part of the neuron plays a role in the communication of information throughout the body.2. Dendrites are treelike extensions at the beginning of a neuron. They increase surface area of the cell body and are covered with synapses. Synapses receive information from other neurons and transmit electrical stimulation to the soma.3. The soma is where the signals from the dendrites are integrated and passed on. The soma and nucleus do not play an active role in the transmission of the neural signal. Instead, they serve to maintain the cell and keep the neuron functional.4. The axon hillock is located at the end of the soma and controls the firing of the neuron. If the total strength of the signal exceeds the threshold limit of the axon hillock, the structure will fire a signal (known as action potential AP) down the axon.5. The axon is the elongated fiber that extends from the cell body to the terminal endings and transmits the neural signal. The larger the axon, the faster it transmits information. Some axons are covered with myelin that acts as an insulator. Myelinated axons transmit information much faster than other neurons. Most neurons have only one axon.6. The terminal buttons (sometimes called synaptic boutons) are located at the end of the neuron and are responsible for sending the signal on to other neurons. At the end of the terminal button is a gap known as a synapse. Neurotransmitters are used to carry the signal across the synapse to other neurons. 6
  7. 7. Opsins and trans-membrane potential
  8. 8. Opsins - transgenic mouseOpsin Wavelength Action Opsin systemsChR2 470 nm Cation channel, depolarization of membrane ChAT-ChR2 transgenic mouse,(Chlamydomonas potential - “Fire” Thy1-ChR2 transgenic mousechannelrhodopsin-2) Lenti-ChR2 virus infected slicesVChR1 (Volvox 535-589 nm Cation channel, depolarization of membrane Thy1-VChR1 transgenic mousechannelrhodopsin-1) potential - “Fire”NpHR (Natronomonas 589 nm Chloride pump, hyperpolarization of Under considerationhalorhodopsin ) membrane potential – “Silence”opto-α1AR 500 nm Light-activated Gq protein, activation of PLC Lenti-OptoGq virus under pathway – Phospholipase C – G-coupled construction receptorsopto-β2AR 500 nm Light-activated Gs protein, activation of AC Lenti-OptoGs virus under pathway construction
  9. 9. Precise and delicate control needs targeted illumination 9
  10. 10. MOSAIC® Patented Digital Mirror Device (DMD). DMD developed by Texas Instruments and is widely used in display devices. Features for Optogenetics Epi lamp adapter • Simultaneous illumination of multipleDMD head complex ROIs for FRAP and photo- activation Laser zoom • Simultaneous Photo-activation/ collimator combined with Electrophysiolgy • Constrained illumination – limit illumination to measurement areas DMD WF e.g. LED
  11. 11. MOSAIC® Spectral Performance M osaic Spectral Throughput 45 40 35 30 Throughput % 25 20 15 10 5 0 300 350 400 450 500 550 600 650 700 750 800 Wavelength (nm)
  12. 12. MOSAIC® On Fixed Stage Microscopes
  13. 13. MOSAIC® Features Simultaneous illumination of multiple ROIs in real time with zero delta time Diffraction limited resolution Spectral range (T > 10%) (365 to 800 nm). Extinction ratio: ~1000:1 Minimum pulse width/exposure time for a single pattern = 75 µs. System frame to frame refresh (a new pattern) is 200 µs or 5kHz frame rate. Unlike projector-based systems Mosaic is NOT controlled by graphics technology… Custom drive electronics delivers precise illumination timing and synchronization Asynchronous external trigger and sync output Custom optical interfaces for most scopes, optical fill and resolution Multi –port optical input available for various illumination sources including: - Lasers and laser diodes – 375, 405, 445, 488 nm - LED e.g. XLED1 – 375-750 nm - Mercury Lamp – 365 nm Hg line - uncaging - Sutter DG4, EXFO X-Cite, Andor AMH- - Others available upon request
  14. 14. Mosaic - Fields of Illumination ZCX2 0.4 mm 04 01 3x fields of ZCX1 0.4 mm Zoom laser 06 illumination Optics ZCX2 0.2 mm ZCX1 0.2 mmPower density, Р ≈ (Ps(λ)*T(λ)*M2)/(AF))Where Ps(λ) = source power at λT((λ) = Mosaic throughput at λM = objective lens magnificationAF = Area of FOI at primary image planeOptical power density required at specimen :•1 to a few 10’s mW/mm2 for Optogenetics• Mosaic delivers this with Lasers, LEDs andlamps. e.g. Channelrhodopsin 2 response vs power density (Augustine lab, Duke Uni)
  15. 15. Mosaic - modes of operation On time (ms)1. Individual 500 Binary or Grey Mask Off time (ms) 100 Upload each and expose Ext Trigger Start or “Bulb”2. Sequence Binary or Grey Masks Upload to head Software start (timed) Ext Trigger (start/advance)3. Sequence “Bulb” mode Upload Binary sequence to head Expose each mask for duration of Ext Trigger pulse width Dark Mask n Dark Mask n+1 Dark Illuminate Illuminate 4. All modes provide “Exposure” output to Gate Source 15
  16. 16. Optogenetics –Axon pCLAMP with Andor Mosaic Case #1 o Mosaic used for photo-stimulation only. o Detection – Axon pCLAMP Electrophysiology o Andor Virtual camera enables Mosaic targeting  Capture video or  “image window copy” from 3rd party application Case #2 o Mosaic provides imaging and photo-stimulation (MetaMorph) o The same light source can be shared for both
  17. 17. Use Case#1 Optogenetics:selective photo-stimulation at 1-20 cell level (granule cell isabout 8-10 um diameter) for electrophysiological recording Selective photo-Axon pCLAMPTM and stimulation withAxoPatch 200B Mosaic®Electrophysiologyrecording Courtesy Yakel Lab, NIEHS
  18. 18. What’s wrong with open field stimulation? Open field light stimulation using configuration from Wang et al Multi-peak response!!Mosaic enables targeted stimulation Want a single – single synaptic response. peak response Courtesy Yakel Lab, NIEHS
  19. 19. Use Case#1 Optogenetics: Mosaic + Axon pCLAMP Andor iQ or Axon AxoPatch 200B MetaMorph + and/or Digidata Axon pCLAMP
  20. 20. Stimulating Action Potentials optogenetics Wavelength encoding Optical Excite different ion pumps stimulus Silence with NpHR Electrical Fire with ChRh2 response Image with Ca++ or other
  21. 21. Andor iQ + Axon pCLAMP Protocols Target Illum- VC Sequence – Mosaic SW or SDK TTL sequencing Trigger– PClamp TTL Select LED – PClamp TTL Intensity – USB2 Optical stimulus Electrical response
  22. 22. Use Case#2 Optogenetics:selective photo-stimulation at 1-20 cell level (granule cell isabout 8-10 um diameter) for calcium imagingOptional Axon pCLAMP Selective photo-Electrophysiology stimulation withrecording Mosaic®Mossy fiber giant boutonsCalcium Imaging Courtesy Yakel Lab, NIEHSFM dye Imaging 22 Aug 22
  23. 23. Use Case #2 – Photo-stimulation and Imaging Video monitor MosaicAndor iQ or MetaMorph + pCLAMP Andor iXon EMCCD – Fast and sensitive Baseline Response Stimulus
  24. 24. Optogenetics and Mosaic – Case #2 • Mosaic “Static” exposure mode – iQ 2.7.1 onwards • Triggering may be Internal or External – option needed Baseline Response Stimulus iQ Adapter needs • External trigger option (IQ 2.8) • Static mode – (IQ 2.7.1) • Asynchronous mode (IQ 2.3 onwards) 24 Aug 22
  25. 25. Optogenetics and Mosaic – Case #2 iQ Protocol example • Shows a series of Static FRAPPA actions • Mask is Static until the next FRAPPA action • At the moment the static action cannot be invoked Static regions for imaging Baseline Timed or Static regions for Stimulus Static regions for imaging Response 25 Aug 22
  26. 26. Andor iQ + Axon pCLAMP Protocols Target Illum- VC Sequence – Mosaic SW or SDK TTL sequencing Trigger– PClamp TTL Select LED – PClamp TTL Intensity – USB2 Optical stimulus Electrical response
  27. 27. Opsins not the only game in town - Hylighter optically active proteinFigure 1. An ionotropic glutamate receptor (top left) and a synthetic photoswitch called Figure 2. MIP image of overlayed confocal stacks HyLighter:GFPMaleimide-Azobenzene-Glutamate (MAG; bottom) is the basis for the light-controlled with non-specific tdTomatoe. Note the cell shows widespreadHyLighter ion channel (right). Images courtesy of Dr.Harald Janovjak. (yellow) presence of HyLighter GFP in the plasma membrane, which can be used to silence the neuron when pulse illuminated with 390nm. Pulse illumination at 500nm inactivates the K+ channel. Hylighter is exciting because it is pulse activated and reversible. Mosaic @ 390nm silences action potentials, @ 500 nm removes inhibition – switching off the K+ pump. Recorded with Axon AxoPatch 200B and pCLAMP Janovjak et al (Nature Neuro 2009) Figure 3. Action potentials triggered by current injections are robustly silenced when HyLighter is activated by 390 nm light (violet bar) and no longer inhibited when deactivated with 500 nm light (green bar).
  28. 28. SEXUAL physiology of C. elegans• Definition: Gubernaculum – “a schlerotic structure that aids in the extension of the male copulatory spicules through the cloaca”.• The spicule is the nematode version of a penis and the cloaca is the single posterior opening which serves the reproductive and excretory tracts.• The gubernaculum muscles control movements of the gubernaculum.• The movements of the copulatory spicules are controlled by the protractor muscles,which help to move the spicule into the cloaca. 28
  29. 29. Calcium sensor (GCaMP) combined with ChRh2Intra-vital “opto-physiology “ in C. elegansTransgenic nematode - ChRh2 expressed in gubernaculum and GCaMP expressed in protractor musclesRed channel – co-expression of mCherryOptical stimulation of ChRh2 in C. elegans gubernaculum muscles (red) modulatescalcium levels (GcaMP fluorescence emission) in the protractor muscles (blue) Total duration 21 seconds Courtesy Luis Rene Garcia, Texas A&M Uni Liu Y. et al PLOS Genetics March 2011 29
  30. 30. Intra-vital “opto-physiology “ in C. elegansMetaMorph running Andor iXon EMCCD, Andor Mosaic with illumination at 470 and 560 nm, 30
  31. 31. Closing remarks Optogenetics is a powerful tool for neuro-physiology Axon pCLAMP integrates readily with Optogenetics Optical patterning with intensity and wavelength control provides newpossibilities in the study of neuronal circuits. Mosaic delivers precise optical patterning and sequencing Mosaic integrates easily with pCLAMP and MetaMorph Andor Technology – Booth # 505 Molecular Devices/Axon Instruments – Booth # 2213 31