Figure 1.

Clustered neuronal cultures and experimental procedure. A, top, Bright field image of a clustered neuronal culture 3 mm in diameter. Dark circular objects are neuronal clusters, and straight filaments are connections. The ablated cluster is boxed in red. Bottom, Corresponding fluorescence image after damage. Healthy clusters appear gray. The ablated cluster, with all its neurons dead, appears bright. Boxed clusters are those whose spontaneous activity is represented in panels E, F. B, Sketch of the multimodal optical system for fluorescence imaging and laser microsurgery. C, Actual field of view in the experiments. Two cultures are simultaneously monitored, with one set as control and the other one as target. The latter is the same culture as in panel A, and the red arrowhead signals the ablated cluster. D, Laser microsurgery. The four snapshots illustrate the action of the laser as it progressively scans the cluster to be ablated, delivering in each step a high energy, high penetration pulse that kills the neurons and vaporizes water. The time interval between panels is 20 s. E, Spontaneous activity before damage for the five clusters highlighted in A. Activity is rich and all clusters fire together in a highly coordinated manner. The red arrowhead marks the cluster to be ablated. F, Corresponding activity after damage, with the ablated cluster completely silent. Its immediate neighbors are initially silent but recover activity after ∼10 min, although with lower firing rates and amplitudes (black arrowheads). Clusters more distant from damage maintain their activity after ablation, although with a reduced firing rate.