Part 2 of 2 of lecture series introducing undergraduate neuroscience students to the core electrophysiological and imaging techniques used to study neuronal activity.

1. Imaging Neuronal Activity
© Stephen P. Currie, University of Edinburgh 2017

2. Learning Intentions
• Gain basic understanding of calcium imaging and 2-photon microscopy
• Learn about the strengths and weaknesses of some common imaging
techniques
• Draw on knowledge of electrophysiological techniques (lecture 1) to
compare and contrast with imaging techniques

3. Limitations of electrophysiology (recap)
Spatial resolution very poor
Difficult to maintain recordings over a long period of time
Monitor activity of identified parts of a neuron
Monitor activity of network/large numbers of cells
Chronic recording of the same cells

4. Imaging Intraneuronal Activity
How to image neuronal activity?
Voltage sensitive dyes exist but not yet sensitive/fast enough for many applications
Ca2+ increases via:
Entry through VGCC & NMDA receptors
EPSP induces release from intracellular stores
Action potentials always visible and most EPSPs visible. Exact potential not
measureable and hard / impossible to comment on subthreshold effects
Reprinted by permission from Macmillan Publishers Ltd: Nature
Inoue et al. copyright (2015)
Genetically-encodedSynthetic dyes
Reprinted by permission from Macmillan Publishers Ltd: Nat.Neuro,
Volterra & Meldolesi , copyright (2005)

5. Imaging Intraneuronal Activity: Challenges
Bits of neurons are really small!
Brain tissue scatters light. Deeper = more scattering
X,Y resolution of fluorescence imaging good enough to see
e.g.: single spines (but not to measure exact dimensions).
Reprinted by permission from Macmillan Publishers Ltd: Nat. Materials, Zagorovsky & Chan copyright (2013)

6. 2-Photon Fluorescence Excitation
S  I2
• Multiple photons must be absorbed
simultaneously, with each providing
only part of the total energy required
• Typically long wavelength near-
infrared (NIR; 700-1000nm) used for
excitation
• There is a non-linear dependence on
excitation intensity
© 2008 Kherlopian et al; licensee BioMed Central Ltd

7. Because of the square intensity relationship
Illumination with one-photon and two-photon wavelengths.
Ryota Homma et al. Phil. Trans. R. Soc. B 2009;364:2453-
2467
© 2009 The Royal Society

8. Two-photon scanning microscopy
Reprinted by permission from Macmillan Publishers Ltd: Nature Svoboda et al. copyright (1997)

9. 2-Photon Imaging of Intracellular Activity
Frequently used in conjunction with whole-cell patch-clamp in vitro and also possible in vivo
Calcium Indicators:
• Can be injected into single cell via patch electrode
• Originally bulk loaded to area of interest (OGB)
• Now usually expressed via virus using genetics (GCaMP etc)
Reprinted by permission from Macmillan Publishers Ltd: Nature Chen et al. copyright (2013)

10. Reprinted by permission from Macmillan Publishers Ltd: Nature Chen et al. copyright (2013)

11. EPSC & calcium signals restricted to single spines
Lur & Higley 2015, Cell Reports

12. Reprinted by permission from Macmillan Publishers Ltd: Nature Matsuzaki et al. copyright (2001)
Uncaging show that larger spines have larger EPSCs
Electron microscopy shows larger spines have more AMPARs
Imaging with single spine-resolution now possible
Reprinted by permission from Macmillan Publishers Ltd: Nature Chen et al. copyright (2013)

13. Hübener 2003, Current Opinion in Neurobiology
Imaging Network Activity in Vivo:
Intrinsic Optical Imaging
• Uses different colour of
oxygenated vs
deoxygenated blood
• More activity = less oxygen
• Spatial resolution very poor
• Signal is tiny: average
many trials
• Equipment cheap, can look
at large areas.

14. Christoph Stosiek et al. PNAS 2003;100:7319-7324
©2003 by National Academy of Sciences
Imaging Network Activity in Vivo: Then…

15. Carsten Tischbirek et al. PNAS 2015;112:11377-11382
©2015 by National Academy of Sciences
Imaging Network Activity in Vivo: Now…

16. © Stephen P. Currie,
University of
Edinburgh 2016
GCaMP6f imaging in layer 2/3 of mouse primary visual cortex

17. Reprinted by permission from Macmillan Publishers Ltd: Nature H Ko et al. copyright (2011)
Imaging functional properties of neurons
in vivo and identifying the same neurons in vitro.
Step 1: in vivo calcium
imaging determines
orientation selectivity of a
large population of cells
Step 2: match in vivo
imaging with in vitro slices

18. Relating orientation and direction preference to connection
probability among L2/3 pyramidal neurons
Step 3: Make in vitro whole-
cell recording from slices
Electrophysiological recording
of synapses
Cells with similar orientation preferences
are more likely to be connected
Such feature-selective connectivity may be
the basis of subnetworks
Reprinted by permission from Macmillan Publishers Ltd: Nature H Ko et al. copyright (2011)

19. Jercog et al. 2016, CSHP
Chronic imaging of the same neurons
Day 1 Day 50
You can study population dynamics during learning
Reprinted by permission from Macmillan Publishers Ltd:
Nature Tian et al. copyright (2009)

20. Bocarsly et al. 2015 , Biomed Optics Express
Next generation of in vivo imaging
3D imaging Imaging deep structures
Gradient Refractive Index (GRIN) lenses
Reprinted by permission from Macmillan Publishers
Ltd: Nature Katona et al. copyright (2012)
Reprinted by permission from Macmillan Publishers Ltd: Nature Kim et al. copyright (2012)

21. Helmchen, Denk & Kerr 2013, CSHP
Terms to look out for:
Anaesthetised
Awake, behaving
Freely moving
Next generation of in vivo imaging
Freely moving

22. MEAs: excellent temporal resolution, poor spatial resolution and stability
Intrinsic optical imaging: cheap and easy but population averages only
2-P Ca imaging: single cell resolution, long-term, moderate temporal resolution
Summary
Network activity
Dendritic/axonal patch clamp: direct voltage recording, difficult, limited spatial resolution
Intrinsic optical imaging: cheap and easy but population averages only
2-P imaging: single-spine resolution, long-term stablility, amplitude almost all-or-none,
structural information.
Intraneuronal Activity

23. Reference List
Bocarsly ME, Jiang WC, Wang C, Dudman JT, Ji N, Aponte Y (2015). Minimally invasive microendoscopy system for in vivo functional imaging of deep
nuclei in the mouse brain. Biomed Opt Express. Oct 23;6(11):4546-56. doi: 10.1364/BOE.6.004546.
Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS
(2013). Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. Jul 18;499(7458):295-300. doi: 10.1038/nature12354. [RightLink #:
4121930571593]
Helmchen F, Denk W, Kerr JN (2013). Miniaturization of two-photon microscopy for imaging in freely moving animals. Cold Spring Harb Protoc. Oct
1;2013(10):904-13. doi: 10.1101/pdb.top078147.
Homma R, Baker BJ, Jin L, Garaschuk O, Konnerth A, Cohen LB, Zecevic D (2009). Wide-field and two-photon imaging of brain activity with voltage-
and calcium-sensitive dyes. Philos Trans R Soc Lond B Biol Sci. Sep 12;364(1529):2453-67. doi: 10.1098/rstb.2009.0084.
Hübener M (2003). Mouse visual cortex. Curr Opin Neurobiol. Aug;13(4):413-20.
Inoue M, Takeuchi A, Horigane S, Ohkura M, Gengyo-Ando K, Fujii H, Kamijo S, Takemoto-Kimura S, Kano M, Nakai J, Kitamura K, Bito H (2015).
Rational design of a high-affinity, fast, red calcium indicator R-CaMP2. Nat Methods. Jan;12(1):64-70. doi: 10.1038/nmeth. [RightLink #:
4121900049651]
Jercog P, Rogerson T, Schnitzer MJ (2016). Large-Scale Fluorescence Calcium-Imaging Methods for Studies of Long-Term Memory in Behaving
Mammals. Cold Spring Harb Perspect Biol. May 2;8(5). pii: a021824. doi: 10.1101/cshperspect.a021824.
Katona G, Szalay G, Maák P, Kaszás A,Veress M, Hillier D, Chiovini B, Vizi ES, Roska, B, Rózsa B (2012). Fast two-photon in vivo imaging with three-
dimensional random-access scanning in large tissue volumes. Nature Methods 9, 201–208. doi:10.1038/nmeth.1851. [RightLink #: 4121960056546]
Kim JK, Lee WM, Kim P, Choi M, Jung K, Kim S, Yun SH (2012). Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of
internal organs in small animals. Nat Protoc. Jul 5;7(8):1456-69. doi: 10.1038/nprot.2012.078. [RightLink #: 4123180645770]
Ko H, Hofer SB, Pichler B, Buchanan KA, Sjöström PJ, Mrsic-Flogel TD (2011). Functional specificity of local synaptic connections in neocortical
networks. Nature. May 5;473(7345):87-91. doi: 10.1038/nature09880. [RightLink #: 4121941340719]
Lur G, Higley MJ (2015). Glutamate Receptor Modulation Is Restricted to Synaptic Microdomains. Cell Rep. Jul 14;12(2):326-34. doi:
10.1016/j.celrep.2015.06.029.

24. Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (2001). Dendritic spine geometry is critical for AMPA receptor expression in
hippocampal CA1 pyramidal neurons. Nat Neurosci. Nov;4(11):1086-92. doi: 10.1038/nn736. [RightLink #: 4121931207168]
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003). In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A. Jun
10;100(12):7319-24. doi: 10.1073/pnas.1232232100.
Svoboda K, Denk W, Kleinfeld D, Tank DW (1997). In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. Jan 9;385(6612):161-
5. doi: 10.1038/385161a0. [RightLink #: 4124641257436]
Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V,
Svoboda K, Looger LL (2009). Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods.
Dec;6(12):875-81. doi: 10.1038/nmeth.1398. [RightLink #: 4121950056981]
Tischbirek C, Birkner A, Jia H, Sakmann B, Konnerth A (2015). Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator. Proc
Natl Acad Sci U S A. Sep 8;112(36):11377-82. doi: 10.1073/pnas.
Volterra A, and Meldolesi J (2005). Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci. Aug;6(8):626-
40. doi: 10.1038/nrn1722. [RightLink #: 4122610976518]
Zagorovsky K, Chan WC (2013). Bioimaging: illuminating the deep. Nat Mater. Apr;12(4):285-7. doi: 10.1038/nmat3608. [RightLink #:
4123200371620]
Reference List
© Stephen P. Currie, University of Edinburgh 2017