Doctoral School of Neuroscience Turin University June 2009 Exploring synaptic circuits with antibodies and confocal microscopy PartII: Principles and applications of confocal microscopy Marco Sassoè Dip. Anatomia, Farmacologia e Medicina Legale Istituto Nazionale di Neuroscienze Torino, Italy

1. The major advantage of a confocal microscope is: The use of laser illumination The elimination of out-of-focus fluorescent light The high resolution The presence of a Z-motor 4. The Airy Unit defines: The size of the pinhole The size of the laser beam The intensity of the laser beam 2. Compared to a conventional epifluorescence microscope a confocal microscope affords: A much better resolution A marginally better resolution The same resolution 5. In a confocal microscope the specimen is: Illuminated point-by-point Illuminated line-by-line Completely illuminated 3. In a confocal microscope the vertical resolution is: The same as the lateral resolution Better than the lateral resolution Worse than the lateral resolution

Laboratory of Synapse Development and Plasticity Annarita Patrizi Laura Viltono Federica Briatore Development of GABAergic synapses: from synaptic adhesion to activity-dependent synaptogenesis Elena Frola Marta Pallotto – Patrizia Panzanelli Synaptic integration of adult-generated neurons Maurizio Giustetto Synaptic plasticity induced by morphine in the mesolimbic pathway Giulio Srubek Tomassy Synapse organization in a mouse model of Rett syndrome Alessandro Ciccarelli Elena Boggio

Synapses: 1014 (100-500 trillion) 1 mm3 of cerebral cortex: one billion synapses!

The Nobel Prize in Chemistry 2008 Roger Y. Tsien Martin Chalfie Osamu Shimomura 8

L’eliminazione genetica di MeCP2 induce alterazioni sinaptiche neurone-specifiche Maurizio Giustetto Elena Boggio VI I strato IV V II-III 100 µm WT KO WT S t r a t o 1 KO WT S t r a t o 5 GFP / NeuN 10

Ruolo dell’inibizione GABAergica nell’integrazione sinaptica dei neuroni neogenerati WT KO Marta Pallotto

Resolution R = 0,5  / NA

GABA GABAARa1 PSD95 Panzanelli et al., 2004 Fritschy et al., 2006 Sassoè-Pognetto et al., 2003

Microscopio a epifluorescenza excitation emission autofluorescence Microscopio a epifluorescenza Indirect (secondary) fluorescence fluorochromes fluorophores

The pinhole is conjugate to the focal point of the lens Sistema confocale Sorgente luce puntiforme Illuminazione puntiforme del campione (con obiettivo ad elevata AN) Pinhole su piano di rilevamento Vantaggi: Eliminazione piani fuori fuoco – immagini più nitide Eliminazione di interferenza laterale – aumento contrasto Sezioni ottiche (e ricostruzioni 3D) Miglior risoluzione (teorico) TUBE LENS The pinhole is conjugate to the focal point of the lens confocal