Imaging NMDA Spike/plateaus in Thin Dendrites in Brain Slices Using Genetically-encoded Calcium Indicators. (360G-Wellcome-083850_Z_07_Z)

Goal: to identify transgenic mice which can be used for imaging NMDA spikes in vivo. Various promising strains, starting with CERTN-L15 (Griesbeck lab, Germany), will be bred and genotyped. Somatosensory cortical brain slices will be cut from individuals with this fluorescent calcium sensing protein. Targetted whole-cell recordings will be made from individual pyramidal neurons expressing the protein, under visual control, using a two-photon microscope (Ultima, Prairie Technologies), already in the lab. Brief, focal pulses of glutamate will be uncaged or iontophoresed onto individual basal, apical oblique and apical tuft dendrites, and NMDA spike/plateaus will be evoked. Glutamate and voltage will be varied to establish thresholds and to quantify variations in fluorescence (calcium) transients with spike/plateau duration, which increases with both glutamate and depolarisation. Fluorescence transients will be mapped along the length of the input dendrite and in neighbouring dendrites , using a number of excitation wavelengths. The input site will be moved up and down single dendrites, and to different dendrites. To mimic in vivo activity, we will test different patterns of glutamate uncaging distributed along the length of input dendrites. Various strains of mouse will be tested, to identify most promising candidates for in vivo imaging.