1. Two photon fluorescence microscopy study of calcium accumulation in cerebellar granule cells Alessandro Esposito 1, Francesca Pellistri 1, Aroldo Cupello 2, C. Marchetti 3 and M. Robello 1 1 INFM, Dipartimento di Fisica, Università di Genova 2 CNR, Istituto di Bioimmagini e Fisiologia Molecolare 3 CNR, Istituto di Cibernetica e Biofisica

2. 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.

3. Quantum Perturbation Theory: Two Photon Microscopy - Theory First not null term: Two Photon Absorption always TPLSM CLSM Null term using IR-NIR light with visible-UV probes Focal plane selection during excitation and not during emission

4. 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

5. 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 Mg 2+.

6. Typical granule cells observed with two-photon microscopy and labelled with Oregon Green BAPTA1 (6 M). The view was captured during NMDA (100 M) stimulation. Rectangles (A,B) are examples of ROIs 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

7. 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 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%

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. In this plot there is also a slow exponential component. KCl 75mM 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

9. Intracellular calcium increase due to NMDA stimuli Stimulation by 100 M NMDA, in the absence of Mg 2+ 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%.

10. 1 1 To get univocal data from neurites upon stimulation with NMDA was very difficult. Depending on the neurites 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

11. Fluorescence increases in cell bodies and neurites after NMDA activation and KCl depolarisation

12. Current neuroscience researches by TPM TPM-PatchClamp combined technique: -calcium current and accumulation -local stimulation by Caged-Coumpounds Immunofluorescence and GABA A receptor: -cytoskeleton correlation -subunits membrane distribution Voltage- and NMDA- activated calcium accumulation in neurites and cell bodies - ratiometric calcium imaging; - Pb 2+ uptake and accumulation analysis by lead quenching Indo-1 and TPM:

13. TPM and neuroscience @ Genoa Francesca Pellistri Camilla Luccardini Mauro Robello Alessandro Esposito Federica Merlo Marzia Pisciotta Thanks to Carla Marchetti and Aroldo Cupello (CNR) Silvia CasagrandeRaffaella Balduzzi