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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

Imaging Sodium Transients as a Response to Local Uncaging of MNI-Glutamate

Author: C. Kleinhans, K. W. Kafitz & C.R. Rose

Institute: Institut für Neurobiologie

Organization: Heinrich-Heine-Universität, Düsseldorf, Germany

 

Whole-cell patch-clamp, multi-photon sodium imaging and (UV)-light-induced uncaging of glutamate were combined to investigate postsynaptic sodium signals in cellular micro domains of central neurons. In detail, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices. In addition, neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines.

Cellular micro domains were perfused with MNI-glutamate. Glutamate was locally uncaged by using a 355 nm pulsed laser. The laser beam (diameter: approx.. 1.5 µm) was directed to the region of interest using the scanner based system UGA- 40.

Focal uncaging of glutamate close to dentritic spines led to observable sodium transients.

 

For more detailed protocol visit JoVE: DOI: 10.3791/52038

Setup:

  • Microscope: Olympus BX51WI/Fluoview 300
  • Objective:    Nikon 60x NIR Apo NA 1.0 water immersion

Rapp OptoElectronic:

  • System:        UGA-40 – point scanning device
  • Light source:            DL-355/10 diode laser
  • Approx. sample spot size:   1.5 µm

Combined techniques:

  • Slice preparation of hippocampus
  • Multi-photon laser scanning microscopy
  • Intracellular sodium imaging
  • Local glutamate perfusion
  • Local uncaging of MNI-glutamate

Figure 1: Sodium signals and synaptic currents induced by flash photolysis of caged glutamate. (A) Maximal projection image of a CA1 pyramidal neuron loaded with SBFI via the patch pipette (PP). The box indicates the area shown enlarged in B. LP indicates the position and orientation of the pipette for local perfusion of caged glutamate. (B) High power maximum projection of a stack of optical sections of a dendrite with adjacent dendritic spines. The stacks of optical sections underwent z-alignment and deconvolution. The red cross indicates the target region of the uncaging beam. The orange dotted line delineates the region of interest from which the fluorescence emission was recorded. (C) Left: Sodium signal (upper row) and somatic inward current (lower row) induced by flash photolysis of caged glutamate (indicated by yellow flash). The red line represents a fit of the experimental data. The grey area represents the period in which the uncaging flash (300 msec) obstructed the imaging of SBFI fluorescence. Right: Without pre-perfusion with caged glutamate, the same UV-flash neither evoked a change in SBFI emission, nor an inward current.

 
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