1. Cortical microcircuit (~1mm 3 ) -Structure: Cell types Connections Random Specific Canonical Areal/Species -Function: Feedforward Feedback

2. Calcium imaging of cortical microcircuits

3. Neuron. 1991 Mar;6(3):333-44. Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters. Yuste R, Katz LC. Laboratory of Neurobiology, Rockefeller University, New York, New York 10021. We assessed the pathways by which excitatory and inhibitory neurotransmitters elicit postsynaptic changes in [Ca2+]i in brain slices of developing rat and cat neocortex, using fura 2. Glutamate, NMDA, and quisqualate transiently elevated [Ca2%]i in all neurons. While the quisqualate response relied exclusively on voltage-gated Ca2+ channels, almost all of the NMDA-induced Ca2+ influx was via the NMDA ionophore itself, rather than through voltage-gated Ca2+ channels. Glutamate itself altered [Ca2+]i almost exclusively via the NMDA receptor. Furthermore, synaptically induced Ca2+ entry relied almost completely on NMDA receptor activation, even with low-frequency stimulation. The inhibitory neurotransmitter GABA also increased [Ca2+]i, probably via voltage-sensitive Ca2+ channels, whereas the neuromodulator acetylcholine caused Ca2+ release from intracellular stores via a muscarinic receptor. Low concentrations of these agonists produced nonperiodic [Ca2+]i oscillations, which were temporally correlated in neighbouring cells. Optical recording with Ca2(+)-sensitive indicators may thus permit the visualization of functional networks in developing cortical circuits.

4. Cortical microcircuit (~1mm 3 ) -Structure: Cell types Connections Random  Specific Canonical Areal/Species -Function: Feedforward  Feedback

5. Function of dendritic spines Most excitatory contacts on spines Some contacts on dendritic shafts Plenty of space on shaft Spines must be key for microcircuit More and longer spines in humans! What is the specific function of the spine?

6. 50 µm 5 µm Imaging living dendritic spines with two-photon microscopy

7. Motility of dendritic spines 5m5m

8. What is the function of the spine?: -connect increase surface,volume shorten wire -chemical isolation learning rules/plasticity but non spiny cells too -electrical? amplification? filtering? digitization? no effect? Need to image voltage in spines!

9.  A Second-order nonlinear scattering Unique Properties of SHG  Interface specific : Centrosymmetric, e.g. bulk liquid  (2) = 0; Non-centrosymmetric, e.g. surface,  (2) ≠ 0.  Spectroscopic: resonance enhancement  Ultrafast time resolution SH response is instantaneous Bulk Medium 1 Bulk Medium 2 Fundamental,  SHG, 2  *not oriented *oriented Second Harmonic Generation (SHG)

10. Summary of Nonlinear Optical Phenomena Normal absorption, reflection, refraction P – Induced polarization in the medium E – Electric field vector of the incident light. SHG, sum and difference frequency generation, linear electro-optic (Pockels) effect, optical rectification, EFISH. Two and Three photon absorption, THG, EFISH, quadratic electro-optic effect, optical Kerr effect, degenerate four wave mixing (DFWM), Self Action (self focusing, self phase modulation), coherent Raman effects (Raman induced Kerr effect, coherent Stokes-Raman scattering (CSRS), coherent anti- Stokes-Raman scattering (CARS))   n) – n th order electric susceptibility.

11. Second Harmonic Generation (SHG) Introduction SHG is purely a scattering process, does NOT involve absorption of photons SHG is coherent process in nature SHG can be enhanced when the energy is near transition resonance

12. Two-photon SHG Why SHG? Surface Selective Electric Field Dependent Ideal technique for optical imaging of membrane voltage (or membrane biochemistry) Electrical field change across plasma membrane when cell fires: ~ 10 8 V/m !!  2 eff =  2 +  3 E DC

13. beam expander (1.2x) and spatial filter TPEF PMT Ti-Sapphire laser Pockels cell Scan box polarizer pinhole Retardation wave plate (  and/or  lamp SHG PMT Ch 1 Ch 2 Fluoview data acquisition

14. Ti-Sapphire laser Pockels cell Scan box polarizer pinhole Retardation wave plate (  and/or  lamp SHG PMT Ch 1 Ch 2 Fluoview data acquisition beam expander (1.2x) and spatial filter TPEF PMT Lock-in Amplifier Function generator EO driver

15. Retinal chemical structure and absorption spectra Biochemistry, Stryer

16. all-trans retinal SHRIMP- Second harmonic retinal imaging of membrane potential

17. Circularly polarized light C293 cells SHGTPEF

18. SHG of hippocampal neurons (all-trans retinal) Z scan 1mm step

19. 25% per 100 mV, flips sign when applied from the inside

20. All-trans retinal loaded neurons from rat hippocampus

22. FM 4-64

23. FM4-64-SHG of layer 5 pyramidal cell in slices mouse V1

24. Voltage sensitivity of SHG (FM4-64 )

25. SHG has large voltage sensitivity (14-25%/100mV)

27. Order parameter: N=5 Neuron SHG depends on chromophore orientation

28. Emitted light’s different polarization components can be isolated: FM 4-64 insert into the membrane with an average angle of ~35 o SHG depends on chromophore orientation

29. Linearly polarized light C293+ kidney cells Composite TPEF (green) SHG (Blue)

30. Circularly polarized light C293+ kidney cells

31. N=3Same cell Chromophore angle does not depend on membrane potential

32. Imaging somatic action potentials: SHG is as fast as measurements

33. 35min (10x, Z) 5m5m Measurements of SHG from Dendritic Spines 50min (3x, Z) 20  m 5m5m

34. (n=6) Soma (n=6)Spine (n=7) Normalized SHG Change (%) p=0.8 (t-test) (n=6) SOMA Spines SHG Changes in Spines by Action Potentials Voltage Change (Soma) SHG Change (Soma) Voltage Change (Soma) SHG Change (Spine)

35. Cable structure: Imaging of Membrane Potential Maps

36. 20% 14% 7% 14% 22% 4% 13% 22% 13% 6% 9% 5% 11% 13% 6% 9% 11% 13% 15% 7% 17% 9% 17% 24% 13% 9% 12% 20% soma 11% 14  m basal 9% 10  m basal 14% 42  m basal branch (3) 17% 30  m basal branch 20% 9  m basal 9% 13  m apical 7% 66  m basal branch (3) 20% 27  m oblique 22% 11  m basal 17% 32  m oblique 14% 24  m basal branch 24% 9  m basal 4% 40  m basal branch (3) 13% 23  m basal branch 13% 63  m oblique 19% 18  m basal 5% 45  m oblique 12% 35  m basal branch (3) 11% 46  m oblique 15% 9  m basal 9% 26  m basal branch 11% 8  m basal 13% 29  m basal branch 6% 24  m basal branch 9% 37  m basal branch 13%

37. SHG imaging of neuronal populations

38. Linearly polarized light C elegans GFP-mec4 SHGTPEF

40. Morphological differences in spines

41. Somatic DC potentials are attenuated in longer spines: The spine neck filters membrane potentials

42. Data: Data1_B Model: Roberto Equation: y=1/(a+b*x) Weighting: yNo weighting Chi^2/DoF= 0.05427 R^2= 0.28877 a0.758870.21487 b0.861520.38357 R = -0.53 Voltage divider: f(x)=1/(a+b*x)

43. Two-photon glutamate uncaging to activate single spines

44. Top and Bottom quartiles of the distribution Uncaging response depends on spine neck length

45. Voltage divider: f(x)=1/(a+b*x) Weighting with StEr Data: Data1_B Model: Roberto Equation: y=1/(a+b*x) Weighting: yw = (data1_c) Chi^2/DoF= 0.00796 R^2= 0.39976 a0.733570.92866 b0.845072.22223 R= -0.62

46. n = 5 (>1.5 µm neck length) Two-photon calcium imaging: long spines activated by glutamate, but silent at the soma Long spines more prevalent in humans voltage calcium

47. 50 ms 1 mV 2 mV Why neck filtering? Role of spines in input summation

48. spines: slope 0.97  0.01; shafts: slope 0.78  0.01; p<0.005, Mann-Whitney). Spines: slope 1.04  0.02; Shafts: slope 0.69  0.02; p<0.005, Mann-Whitney). Spines linearize input summation

49. Linear summation independent of location, distance

50. Imaging of voltage in dendritic trees and spines Imaging of action potentials invading spines Spine neck filters membrane potentials in both directions from soma to spine: SHG from spine to soma: glutamate uncaging Long spines are electrically silent Spine electrically isolates inputs and implement linear summation of inputs Summary:

51. SHG: Boaz Nemet, Jiang Jiang, Mutsuo Nuriya Uncaging: Roberto Araya Ken Eisenthal, Chemistry Dept., Columbia National Eye Institute SHG work was never funded