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ASCB | EMBO San Diego 2018
Annual Meeting of the American Society for Cell Biology and the European Molecular Biology Organization

Dec 08-12, 2018, San Diego, CA, USA
Booth Nr. 1137 (of 89Nort / Chroma Technology)

 

BPS Baltimore 2019
63rd Annual Meeting of the Biophysical Society

Mar 2-6, 2019, Baltimore, Maryland, USA
Booth Nr. 503

 

NWG Göttingen 2019
13th Meeting of the German Neuroscience Society

Mar 20-23, 2019, Göttingen, Germany
Booth Nr. 8

Calcium uncaging - Light induced stimulation of hippocampal neurons

Marcus T. Knopp1, Tobias Bonhoeffer1, U. Valentin Nägerl²

 

1 Max Planck Institute of Neurobiology, Dept of Cellular and Systems Neurobiology, Am Klopferspitz 18, D-82152 Martinsried
² Inserm U862/Université Victor Segalen Bordeaux 2 146, rue Léo Saignat, F-3076 Bordeaux



Recent improvements in light microscopic techniques, in particular laser-scanning fluorescence microscopy, in combination with optical stimulation methods, namely optical switches and caged molecules, have made it possible to study the structure and function of neurons and their synapses in intact brain tissue with high spatial and temporal resolution. These novel techniques are increasingly making it possible to study the neuronal mechanisms underlying brain functions such as learning and memory. 

We have implemented and tested an optical technique based on epi-fluorescence microscopy to study neuronal communication at the level of single synapses. The idea was to be able to dynamically raise the concentration of Ca2+ ions in dendritic spines or presynaptic nerve terminals of neurons, affecting calcium dependent protein systems involved in synaptic transmission and therefore inducing synaptic plasticity. For this, we made use of photo-labile calcium chelators, known as calcium-cages, which rapidly release Ca2+ ions upon the absorption of UV light. Fast CCD camera-based imaging and the use of calcium sensitive dyes made it possible to quantify the UV light-induced changes in the Ca2+ concentrations. After validating the parameters of the experimental setup, including light intensities, various acquisition parameters as well as the concentrations of the calcium-cage and the calcium-dye, these experiments were successfully transferred from the cuvette to organotypic slice cultures of the mouse hippocampus and combined with electrophysiological recordings to show the technique's aptitude for studies in a living neuronal system. 

For future prospects the implemented method should be enhanced by high resolution microscopic techniques, such as two-photon or STED-microscopy, to gain more spatial accuracy in image acquisition at the level of single synapses. Furthermore the spread of the calcium release should be reduced to be able to address smaller cellular structures more directly, and its amount should be increased to mimic natural calcium signals more appropriately.


Figure 1

Figure 1: Optical setup to trigger the flash photolysis of caged calcium and for data acquisition, based on a BX51WI epi-fluorescence microscope (Olympus).
CCD
: CCD camera (Andor iXon)
; DM1, DM2, DM3: dichroic mirrors; EmF: emission filter (500 nm for green, 570 nm for red emission); ExF: excitation filter (460-490 nm for blue, 510-550 nm for green excitation); FOC: fiber optic cable (20 µm diameter); GF1, GF2: grey filters; Obj.: objective (LUMPLFLN 40x/0.8 W, Olympus); Ocu.: ocular; PC: personal computer; PM: positioning mirrors; Sh: shutter; SpC: specimen chamber. Solid black lines represent control cables and data cables, respectively; dashed black lines show the route of the trigger signals.
A
pulsed laser source (DPSL 355/30, Rapp OptoElectronic) was utilized to drive the photolysis, it was coupled into the beam path with a mechanical interface (UGA-40, Rapp OptoElectronic).

Figure 2a

Figure 2a:
Single, light induced stimulation of a neuron. Above the relative change in the fluorescence of a calcium dye (fluo4-FF, Invitrogen) visualizes the uncaging of Ca2+ ions at time point 0; below the corresponding change of the membrane potential is shown. Caged Calcium was brought into the soma of a hippocampal neuron via a patch pipette and was photolysed via a UV flash. The energy delivered to the tissue was modified by the duration of the light flash; it is given in the legend (0.5 ms, 1 ms, 2 ms, 0 ms corresponds to the control measurement).

Figure 2b

Figure 2b:
Successive, light induced stimulation of a neuron.
Above the relative change in the fluorescence of a calcium dye (fluo4-FF, Invitrogen) visualizes the uncaging of Ca2+ ions at time point 0; below the corresponding change of the membrane potential is shown. Caged Calcium was brought into the soma of a hippocampal neuron via a patch pipette and was photolysed via a train of four UV flashes (inter-flash interval: 125 ms). The energy delivered to the tissue was modified by the duration of a light flash; it is given in the legend (0.5 ms, 1 ms, 2 ms, 0 ms corresponds to the control measurement).

 
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