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

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-Universit?t, 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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