Regeneration of functional connectivity within a neural network after different degrees of lesion is of utmost clinical importance. To test pharmacological approaches aimed at recovering from a total or partial damage of neuronal connections within a circuit, it is necessary to develop a precise method for controlled ablation of neuronal processes. We combined an UV laser micro-dissector to ablate neural processes in vitro at single neuron and neural network level with infrared holographic optical tweezers to carry out force spectroscopy measurements. Simultaneous force spectroscopy, down to the sub-pico-Newton range, was performed during laser dissection to quantify the tension release in a partially ablated neurite. Therefore, we could control and measure the damage inflicted to an individual neuronal process. To characterize the effect of the inflicted injury on network level, changes in activity of neural sub-populations were monitored with subcellular resolution and overall network activity with high temporal resolution by concurrent calcium imaging and microelectrode array recording. Neuronal connections have been sequentially ablated and the correlated changes in network activity traced and mapped. With this unique combination of electrophysiological and optical tools, neural activity can be and studied and quantified in response to controlled injury at the subcellular, cellular and network level.