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

-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

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.

Quantum Perturbation Theory:

Two Photon Microscopy - Theory

VHH 0

First not null term: Two Photon Absorption

222 ~IVVw TPE

mminm

always 00 w

TPLSM

CLSM

IVw SPEni 21

Null term using IR-NIR light with visible-UV probes

422

0

22

1

4,,0

z

w

tPtzIn

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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 laserExcitation wavelength: 720nm

Average power at the sample: 5-7mW

Pulsed: 80MHz ; 100-200fsAxial resolution: 700nmLateral resolution: 250nmTime resolution: 3s/pxlTypical used frame time : 100-200ms

Beam path and scan head – NIKON PCM2000

Experimental Setup

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

• 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+.

Typical granule cells observed with two-photon microscopy and labelled with Oregon Green BAPTA1 (6M). The view was captured during NMDA (100 M) 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.

A

B

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 initial value.

KCl 75mM

Analysis of different cell bodies gave an average of 659% (mean value SD) for the fluorescence increase and 92s

for the time constant, in n=20 cells.

The steady state level is 248%

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.

In this plot there is also a slow exponential component.

KCl 75mM

Analysis of different neurites gave an average of 10125% (mean value SD) for the

fluorescence increase and 2.91.5s for the time constant, in n=19 cells.

Some dishes (5) presented also a slow component with a time constant equal to 201s

Intracellular calcium increase due to NMDA stimuli

Stimulation by 100M NMDA, in the absence of Mg2+ and in the presence of 50M 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 6517%.

1

1

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.

2

2

3

3

4

4

Fluorescence increases in cell bodies and neurites after NMDA activation and KCl

depolarisation

Cell FluorescenceNumber Increase

Body 20 65±9%Neurite 19 101±25%Body 18 65±17%Neurite 16 -

KCl

NM

DA

75m

M10

0mM

Plateau Time ConsantBody 24 ± 8 % 9±2sNeurite - 2.9±1.5s (19) / 20±1s (5)

KCl

75m

M

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Thanks to Carla Marchetti and Aroldo Cupello (CNR)

Silvia Casagrande Raffaella Balduzzi