Abstract

We have configured a widefield fast imaging system that allows imaging at 1000 frames per second (512x512 pixels). The system was extended with custom processing tools including a time correlation method to facilitate the analysis of static subcellular compartments (e.g. neuronal varicosities) with enhanced contrast, as well as a dynamic intensity processing (DIP) algorithm that aids in data size reduction and fast visualization and interpretation of timing and directionality in neuronal circuits. This system, together with our custom developed processing tools enables efficient detection of fast physiological events, such as action potential dependent calcium steps. We show, using a specific blocker of nerve communication, that with this setup it is possible to discriminate between a pre and post synaptic event in an all optical way.

© 2014 Optical Society of America

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  1. B. Sakmann and E. Neher, “Patch clamp techniques for studying ionic channels in excitable membranes,” Annu. Rev. Physiol. 46(1), 455–472 (1984).
    [Crossref] [PubMed]
  2. G. Kim and K. Kandler, “Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach,” J. Neurosci. Methods 200(2), 185–189 (2011).
    [Crossref] [PubMed]
  3. J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
    [Crossref] [PubMed]
  4. Y. Y. Ma and D. A. Prince, “Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex,” Neurobiol. Dis. 47(1), 102–113 (2012).
    [Crossref] [PubMed]
  5. J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed., The language of science (Plenum Press, New York, 1995), pp. xxiii, 632, 634 p. of plates.
  6. R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
    [Crossref] [PubMed]
  7. E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
    [Crossref] [PubMed]
  8. K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
    [PubMed]
  9. A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
    [Crossref] [PubMed]
  10. K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
    [Crossref] [PubMed]
  11. G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
    [Crossref] [PubMed]
  12. R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
    [Crossref] [PubMed]
  13. M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
    [Crossref] [PubMed]
  14. D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
    [PubMed]
  15. A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
    [PubMed]
  16. J. B. Furness, The Enteric Nervous System (Blackwell Pub., Malden, Mass., 2006), pp. xiii, 274 p.
  17. W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
    [Crossref] [PubMed]
  18. P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
    [Crossref] [PubMed]
  19. R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
    [Crossref] [PubMed]
  20. F. Helmchen and A. Konnerth, Imaging in Neuroscience: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2011), 1084 p.
  21. S. Schwartz, “Real-Time Laser-Scanning Confocal Ratio Imaging,” Am. Lab. 25, 53–57 (1993).
  22. R. P. Aylward, “The advances & technologies of galvanometer-based optical scanners,” Opt. Scanning: Design Appl. 3787, 158–164 (1999).
    [Crossref]
  23. G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
    [Crossref] [PubMed]
  24. G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
    [Crossref] [PubMed]
  25. K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
    [Crossref] [PubMed]
  26. A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
    [PubMed]
  27. M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
    [Crossref] [PubMed]
  28. J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
    [Crossref] [PubMed]

2014 (1)

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

2013 (2)

R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
[Crossref] [PubMed]

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

2012 (1)

Y. Y. Ma and D. A. Prince, “Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex,” Neurobiol. Dis. 47(1), 102–113 (2012).
[Crossref] [PubMed]

2011 (2)

G. Kim and K. Kandler, “Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach,” J. Neurosci. Methods 200(2), 185–189 (2011).
[Crossref] [PubMed]

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

2010 (1)

K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
[Crossref] [PubMed]

2008 (2)

P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
[Crossref] [PubMed]

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

2005 (3)

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
[Crossref] [PubMed]

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

2002 (2)

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

2000 (3)

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
[Crossref] [PubMed]

1999 (4)

M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
[Crossref] [PubMed]

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

R. P. Aylward, “The advances & technologies of galvanometer-based optical scanners,” Opt. Scanning: Design Appl. 3787, 158–164 (1999).
[Crossref]

1997 (1)

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

1993 (1)

S. Schwartz, “Real-Time Laser-Scanning Confocal Ratio Imaging,” Am. Lab. 25, 53–57 (1993).

1989 (1)

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

1984 (1)

B. Sakmann and E. Neher, “Patch clamp techniques for studying ionic channels in excitable membranes,” Annu. Rev. Physiol. 46(1), 455–472 (1984).
[Crossref] [PubMed]

Antoons, G.

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

Aylward, R. P.

R. P. Aylward, “The advances & technologies of galvanometer-based optical scanners,” Opt. Scanning: Design Appl. 3787, 158–164 (1999).
[Crossref]

Babbey, C. M.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

Bishop-Stewart, J.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Boesmans, W.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
[Crossref] [PubMed]

Brown, K. A.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Camacho, P.

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

Canepari, M.

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
[Crossref] [PubMed]

Capogna, M.

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

Chen, W. N.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Cirillo, C.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

Cohen, L. B.

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Davies, R.

R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
[Crossref] [PubMed]

Duemani Reddy, G.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Dunn, K. W.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

Fain, G. L.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Falk, C. X.

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Fink, R.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Frotscher, M.

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Galligan, J. J.

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

Gee, K. R.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Geiger, J. R. P.

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Gräf, R.

R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
[Crossref] [PubMed]

Graham, J.

R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
[Crossref] [PubMed]

Gray, D.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Hao, M. M.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

Herman, B.

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

Holthoff, K.

K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
[Crossref] [PubMed]

Höpp, H. P.

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Johnson, I.

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Jonas, P.

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Kandler, K.

G. Kim and K. Kandler, “Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach,” J. Neurosci. Methods 200(2), 185–189 (2011).
[Crossref] [PubMed]

Kelleher, K.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Kim, G.

G. Kim and K. Kandler, “Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach,” J. Neurosci. Methods 200(2), 185–189 (2011).
[Crossref] [PubMed]

Konnerth, A.

K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
[Crossref] [PubMed]

Koyano, T.

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

Krasnoperova, N. V.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Lechleiter, J. D.

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

Lem, J.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

LePard, K. J.

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

Li, G.

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

Lindstrom, J.

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

London, J. A.

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Lübke, J.

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Ma, Y. Y.

Y. Y. Ma and D. A. Prince, “Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex,” Neurobiol. Dis. 47(1), 102–113 (2012).
[Crossref] [PubMed]

Martens, M. A.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

Matthews, H. R.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Mazzuoli, G.

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

Michaelis, M.

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

Michel, K.

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

Mubagwa, K.

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

Mueller, K.

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

Neher, E.

B. Sakmann and E. Neher, “Patch clamp techniques for studying ionic channels in excitable membranes,” Annu. Rev. Physiol. 46(1), 455–472 (1984).
[Crossref] [PubMed]

Nelson, M. E.

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

Neunlist, M.

M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
[Crossref] [PubMed]

Nevelsteen, I.

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

Obaid, A. L.

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

Peters, S.

M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
[Crossref] [PubMed]

Prince, D. A.

Y. Y. Ma and D. A. Prince, “Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex,” Neurobiol. Dis. 47(1), 102–113 (2012).
[Crossref] [PubMed]

Publicover, N. G.

R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
[Crossref] [PubMed]

Rietdorf, J.

R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
[Crossref] [PubMed]

Roth, A.

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Saggau, P.

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Sakai, T.

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

Sakmann, B.

B. Sakmann and E. Neher, “Patch clamp techniques for studying ionic channels in excitable membranes,” Annu. Rev. Physiol. 46(1), 455–472 (1984).
[Crossref] [PubMed]

Salzberg, B. M.

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

Sampath, A. P.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Schemann, M.

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
[Crossref] [PubMed]

Schneider, D. A.

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

Schwartz, S.

S. Schwartz, “Real-Time Laser-Scanning Confocal Ratio Imaging,” Am. Lab. 25, 53–57 (1993).

Sipido, K. R.

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

Smith, T. K.

R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
[Crossref] [PubMed]

Stevens, R. J.

R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
[Crossref] [PubMed]

Stewart, R.

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

Tack, J.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
[Crossref] [PubMed]

Takahashi, A.

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

Vanden Berghe, P.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
[Crossref] [PubMed]

Wang, E.

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

Weltens, N.

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

Woodruff, M. L.

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Wu, J. Y.

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Zecevic, D.

K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
[Crossref] [PubMed]

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

Zhou, X.

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

Zimmermann, T.

R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
[Crossref] [PubMed]

Adv. Biochem. Eng. Biotechnol. (1)

R. Gräf, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 57–75 (2005).
[Crossref] [PubMed]

Am. Lab. (1)

S. Schwartz, “Real-Time Laser-Scanning Confocal Ratio Imaging,” Am. Lab. 25, 53–57 (1993).

Annu. Rev. Physiol. (1)

B. Sakmann and E. Neher, “Patch clamp techniques for studying ionic channels in excitable membranes,” Annu. Rev. Physiol. 46(1), 455–472 (1984).
[Crossref] [PubMed]

Cell Calcium (1)

K. R. Gee, K. A. Brown, W. N. Chen, J. Bishop-Stewart, D. Gray, and I. Johnson, “Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes,” Cell Calcium 27(2), 97–106 (2000).
[Crossref] [PubMed]

Front Cell Neurosci. (1)

W. Boesmans, M. A. Martens, N. Weltens, M. M. Hao, J. Tack, C. Cirillo, and P. Vanden Berghe, “Imaging neuron-glia interactions in the enteric nervous system,” Front Cell Neurosci. 7, 183 (2013).
[Crossref] [PubMed]

Gastroenterology (2)

P. Vanden Berghe, J. Tack, and W. Boesmans, “Highlighting synaptic communication in the enteric nervous system,” Gastroenterology 135(1), 20–23 (2008).
[Crossref] [PubMed]

R. J. Stevens, N. G. Publicover, and T. K. Smith, “Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine,” Gastroenterology 118(5), 892–904 (2000).
[Crossref] [PubMed]

J. Auton. Nerv. Syst. (1)

J. J. Galligan, K. J. LePard, D. A. Schneider, and X. Zhou, “Multiple mechanisms of fast excitatory synaptic transmission in the enteric nervous system,” J. Auton. Nerv. Syst. 81(1-3), 97–103 (2000).
[Crossref] [PubMed]

J. Exp. Biol. (1)

A. L. Obaid, M. E. Nelson, J. Lindstrom, and B. M. Salzberg, “Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system,” J. Exp. Biol. 208(15), 2981–3001 (2005).
[Crossref] [PubMed]

J. Microsc. (2)

E. Wang, C. M. Babbey, and K. W. Dunn, “Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems,” J. Microsc. 218(2), 148–159 (2005).
[Crossref] [PubMed]

R. Davies, J. Graham, and M. Canepari, “Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging,” J. Microsc. 251(1), 5–13 (2013).
[Crossref] [PubMed]

J. Neurosci. (3)

D. Zecević, J. Y. Wu, L. B. Cohen, J. A. London, H. P. Höpp, and C. X. Falk, “Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex,” J. Neurosci. 9(10), 3681–3689 (1989).
[PubMed]

A. L. Obaid, T. Koyano, J. Lindstrom, T. Sakai, and B. M. Salzberg, “Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus,” J. Neurosci. 19(8), 3073–3093 (1999).
[PubMed]

G. Li, R. Stewart, M. Canepari, and M. Capogna, “Firing of hippocampal neurogliaform cells induces suppression of synaptic inhibition,” J. Neurosci. 34(4), 1280–1292 (2014).
[Crossref] [PubMed]

J. Neurosci. Methods (1)

G. Kim and K. Kandler, “Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach,” J. Neurosci. Methods 200(2), 185–189 (2011).
[Crossref] [PubMed]

J. Physiol. (4)

K. Michel, M. Michaelis, G. Mazzuoli, K. Mueller, P. Vanden Berghe, and M. Schemann, “Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge,” J. Physiol. 589(Pt 24), 5941–5947 (2011).
[PubMed]

K. Holthoff, D. Zecevic, and A. Konnerth, “Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons,” J. Physiol. 588(7), 1085–1096 (2010).
[Crossref] [PubMed]

G. Antoons, K. Mubagwa, I. Nevelsteen, and K. R. Sipido, “Mechanisms underlying the frequency dependence of contraction and [Ca2+](i) transients in mouse ventricular myocytes,” J. Physiol. 543(3), 889–898 (2002).
[Crossref] [PubMed]

M. L. Woodruff, A. P. Sampath, H. R. Matthews, N. V. Krasnoperova, J. Lem, and G. L. Fain, “Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice,” J. Physiol. 542(3), 843–854 (2002).
[Crossref] [PubMed]

Nat. Neurosci. (1)

G. Duemani Reddy, K. Kelleher, R. Fink, and P. Saggau, “Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity,” Nat. Neurosci. 11(6), 713–720 (2008).
[Crossref] [PubMed]

Neurobiol. Dis. (1)

Y. Y. Ma and D. A. Prince, “Functional alterations in GABAergic fast-spiking interneurons in chronically injured epileptogenic neocortex,” Neurobiol. Dis. 47(1), 102–113 (2012).
[Crossref] [PubMed]

Neurogastroenterol. Motil. (1)

M. Neunlist, S. Peters, and M. Schemann, “Multisite optical recording of excitability in the enteric nervous system,” Neurogastroenterol. Motil. 11(5), 393–402 (1999).
[Crossref] [PubMed]

Neuron (1)

J. R. P. Geiger, J. Lübke, A. Roth, M. Frotscher, and P. Jonas, “Submillisecond AMPA receptor-mediated signaling at a principal neuron-interneuron synapse,” Neuron 18(6), 1009–1023 (1997).
[Crossref] [PubMed]

Opt. Scanning: Design Appl. (1)

R. P. Aylward, “The advances & technologies of galvanometer-based optical scanners,” Opt. Scanning: Design Appl. 3787, 158–164 (1999).
[Crossref]

Physiol. Rev. (1)

A. Takahashi, P. Camacho, J. D. Lechleiter, and B. Herman, “Measurement of intracellular calcium,” Physiol. Rev. 79(4), 1089–1125 (1999).
[PubMed]

Other (3)

J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed., The language of science (Plenum Press, New York, 1995), pp. xxiii, 632, 634 p. of plates.

J. B. Furness, The Enteric Nervous System (Blackwell Pub., Malden, Mass., 2006), pp. xiii, 274 p.

F. Helmchen and A. Konnerth, Imaging in Neuroscience: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2011), 1084 p.

Supplementary Material (2)

» Media 1: AVI (3188 KB)     
» Media 2: AVI (4118 KB)     

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Figures (7)

Fig. 1
Fig. 1 System configuration and noise assessment. (a) Widefield upright microscope equipped with perfusion system, xenon lamp, spritz and electric stimulators, high spatial resolution CCD camera and fast CMOS camera; all automatically controlled through a programmable PC. (b) Average counts over 50 dark frames per image intensifier voltage in steps of 50 only show a sharp increase above 750 of image intensifier voltage. (c) Single dark frame at 1 ms exposure and 850 image intensifier voltage shows (shot) noise very localized in space and random in time. (d) Total noise as standard deviation depends on image intensifier voltage and size of the region of interest. (e) Myenteric ganglion in a preparation dissected from mouse large intestine. Single frame acquired with 1 ms exposure showing high signal-to-noise allowing individual varicosities of neuronal fibers to be resolved in live imaging experiments.

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Fig. 2
Fig. 2 Localization and identification of varicose release sites in a myenteric ganglion dissected from the mouse large intestine. Dashed red lines indicate the region shown in (c) (63x objective). (a) Snapshot images taken with a standard CCD camera (20x objective) yield highly detailed information. Inset Image recorded (standard CCD) with 5x objective shows the ganglionic network and the position of the stimulation electrode on the tissue. (b) Single original frame (20x objective) acquired with 1 ms exposure has sufficient signal-to-noise to resolve individual varicosities of neuronal fibers in live imaging experiments. Arrows indicate active varicosities. (c) Immunohistochemical staining (63x objective) to identify varicosities –synaptotagmin (red arrows), neuronal processes-peripherin (green arrowheads) and neuronal soma-HuC/D (white asterisks). (d-f) Images resulting from our autocorrelation software tool. Output of the autocorrelation tool in a time window of 500 ms before (d) and after (e) the stimulation. (f) Image that results from the subtraction of (d) from (e), which selectively shows those parts of the network that respond within 500 ms after stimulation, clearly highlighting responding varicosities.

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Fig. 3
Fig. 3 Intracellular calcium responses in individual varicosities (top) and transformed signals (bottom). (a) Calcium steps evoked by a serotonin puff. (b) Calcium response evoked by a single electric pulse. (c) Calcium steps evoked by a 20 Hz electric pulse train. (d) Low pass filtered serotonin evoked calcium response (red) and low pass filtered derivative (blue) that is used to automatically detect the stepwise increase. (e) Fourier transform of (b). (f) Fourier transform of (c) confirming a frequency component around 20 Hz.

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Fig. 4
Fig. 4 Dynamic intensity processing (DIP) visualized by input (a,b,c), processing (d,e,f) and output (g,h,i). The input (a) time lapse stack of Fluo4 loaded neuronal cell culture. (b) Time lapse stack of Fluo4 loaded live neuronal tissue. (c) Calcium peaks in these recordings differ only by a few ms, while they are quite distant in space. Therefore, while interpreting the raw data, it is impossible to distinguish when these events occur compared to others. (d) Creating a threshold mask (red) with user defined threshold value. (e) For each non-thresholded pixel: Starting from the time trace of each pixel (upper trace), differentiate (middle trace) and low-pass filter (lower trace) the differential trace. (f) The square of this trace is ideally suited for Igor Pro’s peak detection algorithm (Maximum peaks = 1; Minimum peak percent = 20) that finds the time coordinate of the peak’s maximum. This value is then color coded (using a rainbow color map spread over a user defined time window: from stimulus start until the moment the final responses occur). Pixels with values outside the user defined window are color mapped in black. (g,h) Output of the resulting color coded peak position time value in a new image using the pixel coordinates of the original pixel for either cell culture (g) (Media 1) or nerve tissue (h). Arrows indicate varicosities. Thresholded pixels are not processed and receive a value of 0. (i) The color coded result resolves the spatiotemporal interpretation difficulties of the raw data.

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Fig. 5
Fig. 5 (a) Principle of the expanding rainbow mapping approach. (b) Original single frame (1 ms exposure) in grayscale. (c) Millisecond time gating: selecting individual varicosities from within a complex tissue nerve network that respond in synchrony. (d,e,f) Images selected from an expanding rainbow movie (Media 2). (d) The initial ‘fast’ intracellular calcium wave (arrows) in the ganglion is visualized (electrode bottom right). (e) Expanding the rainbow mapping generates improved color contrast, which emphasizes the newly changed areas while the initial ‘fast’ flux is switched to background blue colors. Arrows indicate varicosities. (f) An image from the end of the response, when the color map is fully stretched, reveals that the ‘late’ fibers run predominantly along the sides of an interganglionic connective (white arrows). Also, varicosities with different timing can be detected (red arrows).

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Fig. 6
Fig. 6 (a) DIP image of a neuronal network in cell culture responding to a single electric pulse. (b) DIP image of the same neuronal network in cell culture responding to a single electric pulse after 2.5 minutes of hexamethonium perfusion. (c) DIP image of the same neuronal network in cell culture responding to a single electric pulse after hexamethonium washout. (d) Schematic representation as a model for (a) of direct activation of one circuit (blue) and activation of a consecutive circuit (green) connected via a cholinergic synapse. (e) Schematic representation as a model for (b) of direct activation of one circuit (blue) and delayed activation of a consecutive circuit (red) with blocked cholinergic synapses. (f) Schematic representation as a model for (c) of direct activation of one circuit (blue) and restored activation of a consecutive circuit (green) again connected via cholinergic synapses.

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Fig. 7
Fig. 7 (a) Double calcium response from an individual varicosity from a myenteric neuron triggered by a single electric pulse. Blue dotted lines indicate automatic step detection for fast analysis. (b) Response from the same varicosity after 2.5 minutes of hexamethonium perfusion. The secondary activation is abolished. (c) The secondary activation reappears after hexamethonium washout. (d) Schematic representation of direct activation and secondary synaptic activation with extended circuit in between. (e) Schematic representation of primary activation and blocked secondary synaptic activation. (f) Schematic representation of primary activation and recovered secondary synaptic activation after hexamethonium washout.

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Equations (1)

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V=  ∑ T=0 M−1 ( ∑ t=0 N−1 (f( t ).f( t+T ))       M

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