Trauma in Neonatal Acute Brain Slices Alters Calcium and Network Dynamics and Causes Calpain-Mediated Cell Death

Preparing acute brain slices produces trauma that mimics severe penetrating brain injury. In neonatal acute brain slices, the spatiotemporal characteristics of trauma-induced calcium dynamics in neurons and its effect on network activity are relatively unknown. Using multiphoton laser scanning microscopy of the somatosensory neocortex in acute neonatal mouse brain slices (P8-12), we simultaneously imaged neuronal Ca²⁺ dynamics (GCaMP6s) and cytotoxicity (propidium iodide or PI) to determine the relationship between cytotoxic Ca²⁺ loaded neurons (GCaMP-filled) and cell viability at different depths and incubation times. PI⁺ cells and GCaMP-filled neurons were abundant at the surface of the slices, with an exponential decrease with depth. Regions with high PI⁺ cells correlated with elevated neuronal and neuropil Ca²⁺ The number of PI⁺ cells and GCaMP-filled neurons increased with prolonged incubation. GCaMP-filled neurons did not participate in stimulus-evoked or seizure-evoked network activity. Significantly, the superficial tissue, with a higher degree of trauma-induced injury, showed attenuated seizure-related neuronal Ca²⁺ responses. Calpain inhibition prevented the increase in PI⁺ cells and GCaMP-filled neurons in the deep tissue and during prolonged incubation times. Isoform-specific pharmacological inhibition implicated calpain-2 as a significant contributor to trauma-induced injury in acute slices. Our results show a calpain-mediated spatiotemporal relationship between cell death and aberrant neuronal Ca²⁺ load in acute neonatal brain slices. Also, we demonstrate that neurons in acute brain slices exhibit altered physiology depending on the degree of trauma-induced injury. Blocking calpains may be a therapeutic option to prevent acute neuronal death during traumatic brain injury in the young brain.