20020911 Trento SIBPA2002 by 9Poq16Eo

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									Two photon fluorescence microscopy study
  of calcium accumulation in cerebellar
              granule cells

        Alessandro Esposito1, Francesca Pellistri1,
    Aroldo Cupello2, C. Marchetti3 and M. Robello1


    1   INFM, Dipartimento di Fisica, Università di Genova
    2   CNR, Istituto di Bioimmagini e Fisiologia Molecolare
    3   CNR, Istituto di Cibernetica e Biofisica
NMDA- and Voltage- activated calcium accumulation
             in neurites and cell bodies


    Our aim was to analyse the calcium distribution and its time
   variations due to NMDA- and Voltage- activated ion channels.
      Ca2+ is a secondary messenger; it mediates signals in the
                            cytoplasm.



                     -confocal imaging is the best choice for its high
                     spatial resolution but it presents a low time
                     resolution
                     -to improve the frame time we can scan little
                     regions of interest
                     -to reduce photobleaching we can use two photon
                     excitation
                        Two Photon Microscopy - Theory

Quantum Perturbation Theory:               H  H0 V
w0   0               always

  1
w  Vni   SPE I
                    2

                                                                            CLSM
  Null term using IR-NIR light with visible-UV probes
                                             ~2
w   VnmVmi
  2 
                                       TPE I
                                 2
                                                                            TPLSM
                m
     First not null term: Two Photon Absorption

                                           4 P 2 t 
ne  I 0, z, t  
            2
                                                                  z   4

                                 w 1    
                                       0
                                                 z
                                                     zR
                                                          2   2


         Focal plane selection during excitation and not during emission
                      Two Photon Microscope


- good focal plane selection;
- reduced photobleaching;
- good resolution;

                                 Ti:Sapphire ROD lightened by the green Millennia laser
Excitation wavelength: 720nm
Average power at the sample: 5-7mW
Pulsed: 80MHz ; 100-200fs                   Axial resolution: 700nm
                                            Lateral resolution: 250nm
                                            Time resolution: 3s/pxl
                                            Typical used frame time : 100-200ms




                                Beam path and scan head – NIKON PCM2000
                        Experimental Setup
• The control solution contained (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0
MgCl2, 10 Glucose, 5 Hepes, pH~7.4 (NaOH).
• The high potassium solution (KCl 75 mM) was prepared by equimolar
substitution of NaCl.
• The response to NMDA was measured in 100 M NMDA and 50 M
glycine, in the absence of Mg2+.

                                   Neurons were incubated in 6 M of the
                                   cell-permeant AM ester form of Oregon
                                   Green BAPTA1for 40-45 min at 37°C,
                                   then washed several times with standard
                                   saline at room temperature. Cultures were
                                   transferred to a recording chamber where
                                   they were continuously superfused with
                                   solutions fed by gravity (3 ml/min).

                                   KCl and NMDA response was acquired in
                                   the same setup condition onto somata
Typical granule cells
observed with two-photon
microscopy and labelled
                                          A
with Oregon Green
BAPTA1 (6M). The view
was captured during
NMDA (100 M)                                    B
stimulation. Rectangles
(A,B) are examples of
ROI’s in which
experimental acquisitions
were performed.



Single optical section by TPE (700nm-250nm axial and lateral resolution); image
  is filtered by a Wiener and a Gaussian (variance 2 pxl) filter both with a 3x3
                          Kernel and then thresholded.
 Intracellular calcium increase due to depolarisation stimuli



                                          Depolarisation caused the
                                          intracellular calcium to increase
                                          and then to decay to a steady-
                                          state level. Washing with
                                          standard solution let the
                                          cytoplasmic calcium
                                          concentration come back to the
              KCl 75mM
                                          initial value.




Analysis of different cell bodies gave an average of 659% (mean value  SD)
 for the fluorescence increase and 92s for the time constant, in n=20 cells.
                       The steady state level is 248%
As concerns the neurites, perfusion
with high potassium resulted in a
sharp peak of fluorescence increase
followed by a decrease to the control
level; this process was complete
before the end of the high potassium
perfusion. Even considering different
parts of the same neurite the shape
of the response did not change.
                                                       KCl 75mM
In this plot there is also a slow
exponential component.


          Analysis of different neurites gave an average of 10125%
         (mean value  SD) for the fluorescence increase and 2.91.5s
                      for the time constant, in n=19 cells.
         Some dishes (5) presented also a slow component with a time
                           constant equal to 201s
Intracellular calcium increase due to NMDA stimuli


                                   Stimulation by 100M NMDA,
                                   in the absence of Mg2+ and in
                                   the presence of 50M glycine,
                                   resulted in the typical
                                   behaviour with a sustained
                                   increase of cell body
                                   fluorescence lasting from the
                                   beginning to the end of the
                                   NMDA treatment.




          Several cells were studied (n=18) with an
         average fluorescence increase of 6517%.
                                   3
                                   1                    24




         1
         2
         3
         4




To get univocal data from neurites upon stimulation with NMDA was very
difficult. Depending on the neurite’s regions which were selected, the
responses to stimulation could vary much. Every neurite was scanned
through various small spatial windows of a few microns each and the
results were very different in the various regions.
Other analysis are more homogeneous but it seems to be related to the
selection of the experimental region on the neurites.
  Fluorescence increases in cell bodies and neurites after
        NMDA activation and KCl depolarisation

                             Cell Fluorescence
                            Number Increase
                    Body      20     65±9%
       100mM 75mM
KCl




                    Neurite   19   101±25%
                    Body      18    65±17%
NMDA




                    Neurite   16        -




                                                      Plateau      Time Consant
                                             Body    24 ± 8 %           9±2s
                                      75mM
                                KCl




                                             Neurite     -    2.9±1.5s (19) / 20±1s (5)
Current neuroscience researches by TPM

 Voltage- and NMDA- activated calcium accumulation
       in neurites and cell bodies

  Indo-1 and TPM:
       - ratiometric calcium imaging;
       - Pb2+ uptake and accumulation
         analysis by lead quenching

  Immunofluorescence and GABAA receptor:
      -cytoskeleton correlation
      -subunits membrane distribution

  TPM-PatchClamp combined technique:
      -calcium current and accumulation
      -local stimulation by Caged-Coumpounds
           TPM and neuroscience @ Genoa


                                    Mauro Robello


              Francesca Pellistri
                                          Alessandro Esposito

                     Camilla Luccardini

            Silvia Casagrande                 Raffaella Balduzzi

  Marzia Pisciotta
                                Federica Merlo

Thanks to
Carla Marchetti and Aroldo Cupello (CNR)

								
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