eNeuro. 2024 Jun 17:ENEURO.0007-24.2024. doi: 10.1523/ENEURO.0007-24.2024. Online ahead of print.

ABSTRACT

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.Significance statement This is the first study to characterize the spatiotemporal dynamics of neocortical injury in acute neonatal slices, mimicking severe penetrating traumatic brain injury, using PI labeling and elevated neuronal Ca²⁺ load as markers for cytotoxicity. We found depth- and time-dependent neuronal damage, leading to altered neuronal responses. Elevate neuronal Ca²⁺ and cytotoxicity were mitigated by pharmacologically inhibiting calpains, a family of Ca²⁺-dependent proteases involved in multiple cell death mechanisms. Our study provides evidence for injury-dependent neuronal and circuit function alterations in neonatal acute brain slices. Calpain inhibition decreased trauma-induced cell death in the neonatal brain, identifying them as potential therapeutic targets at this age.

PMID:38886064 | DOI:10.1523/ENEURO.0007-24.2024