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.


Introduction
Acute brain slices are widely used in neuroscience and have been instrumental in gaining insights into neuroglial function at molecular, synaptic, and cellular levels.Brain slices are well suited for various experimental approaches like electrophysiology, fluorescent imaging, and biochemical and histological techniques (Yamamoto and McIlwain, 1966;Collingridge, 1995;Colbert, 2006).However, the preparation and incubation of the brain slices result in trauma and incubation-dependent cellular injury, mimicking penetrating brain injury.Here, we evaluated the spatiotemporal dynamics of cell death, its correlation with neuronal Ca 2+ overload, and the role of calpains in neonatal neocortical brain slices.
Multiple studies on cultured cells report that injured neurons with high Ca 2+ load often exhibit abnormal nuclear accumulation of otherwise cytosolic GCaMP (Bano et al., 2010;Strasser et al., 2012).Also, Ca 2+ -activated and calpain-mediated processes increase neuronal nuclear membrane permeability, facilitating nuclear translocation of cytosolic GCaMP6s (Bano et al., 2010;Suryavanshi et al., 2024).This nuclear accumulation and Ca 2+ overload impart a "filled" appearance to the injured neuron (Yang et al., 2018).However, contributions of [Ca 2+ ]/calpain-mediated processes in trauma-related injury of acute brain slices remain unexplored, especially in the neonatal brain.
In this manuscript, we evaluated the relationship between trauma-induced Ca 2+ load in neurons and the neuropil with cell death across different tissue depths in acute neonatal brain slices and during several incubation time points.Additionally, we investigated the role of calpains, a family of Ca 2+ -dependent proteases, in this process.We used high-resolution, dualcolor, multiphoton imaging of acute neonatal brain slices expressing a genetically encoded neuronal Ca 2+ indicator (GCaMP6s), which were treated with propidium iodide (PI), a widely used indicator of cell death (Brana et al., 2002;Buskila et al., 2014), to acquire simultaneously neuronal Ca 2+ dynamics and a cytotoxicity profile.Our results demonstrate a depthdependent occurrence of both Ca 2+ overload and cell death, exacerbated by prolonged incubation.This Ca 2+ overload resulted in altered physiology, where neurons with Ca 2+ overload do not respond to stimulus-evoked or seizure-evoked network activity.Using pharmacology, we show that calpain-mediated processes activated during incubation contribute to trauma-induced cell death over time.Thus, our results establish thresholds for imaging noninjured tissue and provide spatiotemporal characteristics and a mechanism of trauma-induced injury in the neocortex of acute neonatal brain slices.

Materials and Methods
Study design and animals.All experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee at The University of Iowa.We used transgenic mice expressing a genetically encoded Ca 2+ indicator, GCaMP6s, under the Thy1 promoter [C57BL/6J-Tg (Thy1-GCaMP6s) GP4.3Dkim/J, Jackson Laboratory, strain #024275].Acute brain slices were prepared at postnatal days 8 to 12 (P8-12) using hemizygous and homozygous, 9 male and 5 female Thy1GCaMP6s mouse pups.We used a total of 27 slices (8 pups) that were incubated in artificial cerebrospinal fluid (aCSF) alone, 16 slices (4 pups) incubated in the calpain inhibitor MDL-28170 (MDL), and 12 slices (3 pups) incubated in the calpain-2-specific inhibitor (Cal 2-1).We used eight staurosporinetreated and five aCSF-treated slices (two pups) to evaluate staurosporine-induced cell death.All the mice were housed in a temperature-and humidity-controlled vivarium with ad libitum access to food and water on a 12 h light/dark cycle.
Preparation of acute brain slices.Mouse pups were anesthetized using isoflurane inhalation and decapitated.Each brain was removed and placed in ice-cold, aCSF containing the following (in mM): 116 NaCl, 3.3 KCl, 1.3 CaCl 2 , 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 10 D-glucose, and 20 D-mannitol along with a high MgCl 2 concentration (2 mM) and kynurenic acid (2 mM) to block glutamatergic receptors (osmolarity, ∼300 mOsm).The aCSF was saturated with carbogen (95% O 2 and 5% CO 2 ) to maintain a pH of 7.3-7.4.Thick coronal sections (450 µm, commonly used at this age) containing the sensory neocortex were cut using a vibratome (Leica VT1000S, new blade used after every single use) while submerged in ice-cold aCSF, as described previously (Leinekugel et al., 1997;Dzhala et al., 2012;Glykys et al., 2019;Takezawa et al., 2023).We also cut 350 µm slices (common adult thickness) for thickness comparison experiments.The brain slices were placed in an interface holding chamber containing aCSF, devoid of kynurenic acid and with 1.3 mM MgCl 2 , at room temperature for 30 min.Next, the temperature was slowly increased to and maintained at 30°C (recovery time).Per prior protocols, the slices were incubated for at least 1 h after sectioning before being transferred to the recording chamber (Glykys and Mody, 2007;Glykys et al., 2009;Takezawa et al., 2023).

Pharmacological interventions.
See below, under Reagents, for a detailed description of pharmacological agents and other chemicals used in this study.Depending on their solubility, stock solutions were prepared for all compounds in dimethyl sulfoxide (DMSO; Sigma-Aldrich # 472301) or regular aCSF.The stocks were then diluted to desired concentrations in aCSF before perfusion.Whenever appropriate, vehicles contained identical DMSO concentrations to respective stock solutions of the drugs (all ≤0.05%).All solutions (vehicle and drugs) were maintained at room temperature and perfused at ∼2 ml/min through a line heater.The solutions took <30 s to reach the recording chamber.To determine the contribution of calpain-mediated processes in cell death, neonatal slices were either incubated in a nonspecific calpain inhibitor MDL-28170 (30 µM, 1-5 h), calpain-2-specific inhibitor Cal 2-1 (0.5 µM, 2-5 h), or vehicle (1-5 h), following 30 min of initial recovery in regular aCSF.Before multiphoton imaging, individual slices were treated with PI (10 µM for 15-30 min).To estimate staurosporine-induced cell death, all slices were incubated either in staurosporine or vehicle for 4 h in a submerged chamber (5 ml volume) saturated with carbogen before PI treatment and multiphoton imaging.Seizure-like activity was generated in acute brain slices by applying 4-aminopyridine (4-AP) at 100 µM, ∼20 min before starting the recordings (Chesnut and Swann, 1988;Perreault and Avoli, 1991;Langton et al., 2022;Liddiard et al., 2024).
Multiphoton imaging.Slices were placed in a submerged chamber constantly perfused with recording aCSF bubbled with carbogen and maintained at 30°C.The location of the sensory neocortex was determined using epifluorescence.Two-photon laser scanning microscopy was performed using the Bruker Ultima galvo-resonant system mounted on an Olympus BX51WIF upright microscope with a water immersion objective (20×, 1.0 N.A.).A single Ti:sapphire tunable laser (Mai Tai HPDS; Spectra-Physics) was used for two-photon excitation.Different excitation wavelengths, ranging from 860 to 1,040 nm, were tested for dual-color acquisition of GCaMP6s and PI emissions.An excitation wavelength of 920 nm was selected as it was optimal for Ca 2+ -dependent excitation of GCaMP6s and generated a 50% peak signal of PI (Extended Data Fig. 1-1A).Scanning was performed with galvo-mirrors.Emitted light was bandpass filtered at 565 nm using a dichroic mirror (T510lpxrxt, Chroma), and green and red emission wavelengths were isolated using specific filters: 525/35 nm (green) and 595/25 nm (red).Two GaAsP or multi-alkali photomultiplier tubes (PMT, Hamamatsu Photonics) were used to simultaneously acquire green and red signals.Three-dimensional stacks (3D) of raster scans were acquired at a 512 × 512 pixel resolution, beginning at the top of the slices to a depth of 300 µM (step size, 2-3 μm).The acquired images contain multiple layers of the sensory neocortex.All images were acquired at 1× digital zoom.The laser power was linearly increased throughout the stack acquisition (16-109 mW from top to bottom) to offset the scattering/absorption of photons in deeper tissue.
Image processing and segmentation.The 3D stacks acquired using two-photon laser scanning microscopy were split into two channels (green and red), converted to 8 bit TIFF format, and processed separately.The Z-stacks were background subtracted and smoothened (median filter; radius, 2), and maximum intensity projections (MIPs) were generated for every five images, each representing a 10-15 μm deep section of brain tissue.To detect GCaMP-filled and PI + cells, a threshold of five times the background's standard deviation (SD) was used to apply a binary mask over MIPs acquired from the green and red channels, respectively.GCaMP-filled and PI + cells were then segmented using the "analyze particles" function (ImageJ) using specific filters (size for PI + cells, 10-100 μm; size for GCaMP-filled cells, 20-200 μm; circularity, 0.5-1 for both).To eliminate false PI + cell detections due to the green bleed-through signal, a subset of PI + cells were manually detected (n = 284 cells), and red/green ratios for each detection were calculated.A Gaussian function was fitted to their frequency distribution to obtain the mean and SD (0.3 ± 0.7).A threshold for red/green intensity ratios obtained from the test dataset (mean minus 1 SD: −0.4) was used to eliminate false red detections from the experimental dataset acquired with automated selection (n = 4,087 red/green ratios; Extended Data Fig. 1-1B).A custom-built macro automatically updated the ROIs representing GCaMP-filled/PI + detections to their corresponding z-planes.To determine GCaMP-filled/PI + colocalization, the centroids of the ROIs detected using the "red" and "green" stacks were matched using a custom-made macro in Igor Pro (WaveMetrics).
To measure the neuronal Ca 2+ transients, raw images in time series were background subtracted, smoothened (median filter; radius, 2), and compressed into MIPs.Circular ROIs representing neuronal somas and concentric, doughnut-shaped ROIs representing the neuropil were generated on the MIPs using a semiautomated, custom-built macro.The neuropil fluorescence was subtracted from the corresponding somatic fluorescence for each frame.To determine the characteristics of evoked Ca 2+ responses, we performed baseline subtraction and normalization (ΔF / F 0 ) of the neuropil-subtracted Ca 2+ signal for each neuron using the following equation: (F t − F 0 ) / F 0 , where F t is the fluorescence intensity of a given frame and F 0 is the average baseline fluorescence.
Reagents.All aCSF solutes, kynurenic acid, PI, 4-AP, and MDL-28170 (MDL) were obtained from Sigma-Aldrich.Staurosporine was obtained from Tocris.Calpain-2-specific inhibitor Calpain 2-IN-1 (Cal 2-1) was obtained from MedChemExpress.PI was diluted from an aCSF stock solution (1 mg/ml, 1,000×) to a final 1 μg/ml concentration.MDL was diluted from a DMSO stock solution (60 mM, 2,000×) to a final concentration of 30 μM. 4-AP was diluted from a stock prepared in water (100 mM, 1,000×) to a final concentration of 100 μM.Cal 2-1 was diluted from DMSO stock solution (5 mM, 10,000×) to a final concentration of 0.5 μM.Staurosporine (100 μg) was dissolved in DMSO to make a 5 mM stock (42.9μl), which was then dissolved in 10 ml aCSF to obtain a final concentration of 20 μM.All stock solutions were stored at −20°C and used within 3 months of preparation.
Statistics.The total number of (or % normalized) PI + and GCaMP-filled cells were calculated at different depths (from 0 to 300, 30 μm steps containing 2-3 MIPs) for multiple slices and plotted as a normalized fraction or total number across a bin of 30 μm.Exponential decays were fitted to the % normalized cells to obtain decay constants (τ).To measure neuropil GCaMP6s intensity across depth, median fluorescence intensities for each 30 μm step were plotted.To determine the effect of incubation time, the number or % normalized cells versus depth plots were generated for slices grouped according to a range of incubation durations (0-1, 1-2, 2-3, 3-5 h) before fitting the exponential decay functions.The normality of distributions was determined using the Shapiro-Wilk test and QQ plots.Statistical tests for parametric data were used for normal distributions, while lognormal distributions were log-transformed before applying parametric tests.Normally distributed data are presented as mean ± 95% confidence interval (CI).Nonparametric data are presented as median ± interquartile range (IQR).Data representing multiple depths within the same perturbation were analyzed using repeatedmeasures one-way ANOVA with Dunnett's test for multiple comparisons.Unpaired parametric data across various time points were analyzed using one-way ANOVA.Data representing multiple depths were compared across groups (or perturbations) using two-way ANOVA with Tukey's or Sidak's post hoc tests, depending on the number of multiple comparisons.Data points representing individual slices or cells were compared across groups using a two-tailed, unpaired t test (parametric data) or Mann-Whitney U test (nonparametric data).For statistical correlations, data were normalized to slice total (PI + and GCaMP-filled cells) or slice maximum value (median neuropil GCaMP6s intensities).To determine a correlation between median neuropil intensities obtained from XY grids and PI + cells, the grids with zero PI + cells were eliminated from the analysis.Spearman's correlation (nonparametric data) was used to identify a statistically significant correlation.Statistical significance was considered at p < 0.05.For detailed statistics, see Table 1.

Results
Evidence of abundant cell death and neuronal Ca 2+ overload on the surface of acute brain slices decreasing exponentially with depth First, we evaluated the anatomical localization of trauma-and incubation-induced cell death and Ca 2+ overload (GCaMP-filled neurons) in the neocortex of neonatal brain slices.We acquired high-resolution, dual-color multiphoton 3D scans (0-300 µm; step size, 2-3 µm) of the somatosensory region from acute Thy1-GCaMP6s slices (Chen et al., 2012) labeled with PI (Buskila et al., 2014), a known marker for cell death (Fig. 1A).We then obtained MIPs for every 10-15 µm step from the 3D stacks (Fig. 1B).These MIPs, representing progressive depths, were used to detect GCaMP-filled neurons and PI + cells.False-positive detections of the PI signal were eliminated using a ratiometric and threshold-based approach (Materials and Methods; Extended Data Fig. 1-1).The numbers of PI + cells and GCaMP-filled neurons were highest in the superficial layers (0-30 µm) and decreased with increasing depth (Fig. 1C).To determine the precise decay rates across depth, we fitted single exponentials to the percent normalized PI + and GCaMP-filled cell counts (Fig. 1D).Both had a similar rate of decay (τ PI + , 41 ± 10 µm; τ GCaMP-filled , 65 ± 25 µm) with ∼75% of average PI + and GCaMP-filled cells in the top 0-90 and 0-100 µm of the slice, respectively.We also found fewer colocalized puncta (GCaMP-filled and PI + neurons) with increasing depth (Fig. 1E; ∼79% colocalized neurons were in the uppermost 30 µm).There was also a significant correlation between cell death (PI + ) and GCaMP-filled neurons across tissue depths (Fig. 1F).Thus, these data indicate a depth-dependent co-occurrence of cell death and elevated neuronal Ca 2+ .
To confirm the efficacy of PI labeling and the segmentation of PI + cells, we induced apoptosis in acute neonatal brain slices by treating them with staurosporine (20 µM for 4 h; Hanse and Gustafsson, 1994;Zhou et al., 2001).We acquired dual-color images from staurosporine-treated and aCSF-treated (control) brain slices.PI + cells and GCaMP-filled neurons were present at different depths across the slice (Extended Data Fig. 1-2A,B).Staurosporine significantly increased the number of PI + cells at various depths compared with aCSF, with a significant increase in decay constants and total PI + cells per slice (Extended Data Fig. 1-2C-E).Staurosporine also significantly increased colocalized puncta across different tissue depths (Extended Data Fig. 1-2F).These results validate our PI labeling and cell segmentation approach.Next, we tested if PI + cells and GCaMP-filled neurons were localized to specific cortical layers.Although the density of PI + cells and GCaMP-filled neurons was slightly higher in deeper layers, the difference between deeper and superficial layers was not statistically significant (Extended Data Fig. 1-3).We then tested if slice thickness and postnatal age subdistribution altered trauma-related cell death and Ca 2+ overload in acute brain slices.We found that thinner slices (350 µm, common thickness used in adult brain slices) had a significant increase in PI + cells and GCaMP-filled neurons at a depth of 270-300 µm, near the opposite surface of the slice (Extended Data Fig. 1-4A,B).We did not observe a postnatal age-dependent effect (P8-9 vs P10-12) on the distribution of PI + cells and GCaMP6-filled neurons across depth (Extended Data Fig. 1-4C,D).
Cell death and elevated neuropil Ca 2+ signal are correlated Elevated Ca 2+ signal in the neuropil is often associated with injury (Berridge et al., 2003).We examined the relationship between cell death and neuropil Ca 2+ signal across different depths in acute neonatal brain slices (Fig. 2A).Like PI + cells, the normalized median neuropil intensities displayed an exponential decrease with depth (Fig. 2B).The normalized number of PI + cells and median neuropil GCaMP intensities acquired from individual MIPs showed a significant correlation (Fig. 2C).Next, we evaluated the correlation between neuropil GCaMP intensities and the number of PI + cells in 2D space (x-y dimensions).We found that PI + cells and median neuropil Ca 2+ signals, measured from individual ROIs within a 4 × 4 grid, showed a significant correlation (Fig. 2E), indicating that PI + cells segregate with elevated Ca 2+ in the neuropil.
Altered responses of GCaMP-filled neurons during evoked and seizure-induced activity During slice preparation, traumatic injury to neurons and their processes can significantly alter neuronal and synaptic physiology (Kirov et al., 1999;Dzhala et al., 2012).Therefore, we evaluated the response of GCaMP-filled neurons during induced network activity.These neurons were characterized by tonically high fluorescence and a lack of Ca 2+ transients during external stimuli (125 µM NMDA puff, 100 ms) or during seizure-like activity induced by 100 µM of 4-AP (Fig. 4; Movies 1, 2).None of the identified filled neurons responded to NMDA puffs or to 4-AP seizure evoked activity (total 39 neurons from 7 slices).
Next, since the superficial tissue is more susceptible to traumatic injury during slice preparation, we tested if seizure-related Ca 2+ dynamics in superficial neurons were altered compared to neurons in deeper tissue.Seizure-like activity was induced by applying 4-AP (100 µM), and neuronal Ca 2+ transients were imaged at different depths (20 or 80-100 µm from the surface; Fig. 5A).4-AP application induced synchronous, transient neuronal Ca 2+ activity in the superficial and deep tissue (Fig. 5B, Movie 3).However, the synchronous neuronal Ca 2+ transients had significantly higher frequencies, amplitudes, and areas under curve in the deeper tissue than the superficial ones (Fig. 5C-F).These results show that GCaMP-filled neurons have altered physiology and modified the network activity.

Calpain inhibition during acute brain slice incubation mitigates cell death
Our results thus far suggest a functional relationship between trauma and cell death and elevated neuronal Ca 2+ .Persistently high intracellular Ca 2+ levels can activate calpains, a family of Ca 2+ -dependent proteases mediating various cellular functions ranging from synaptic plasticity to cell death (Baudry and Bi, 2016;Cheng et al., 2018).Previous findings in cultures suggest that Ca 2+ -dependent calpain activity following neuronal hyperexcitability alters nuclear permeability (Bano et al., 2010;Yamashita et al., 2017), which can result in the translocation of cytoplasmatic GCaMPs into the nucleus, rendering neurons "GCaMP-filled" (Suryavanshi et al., 2024).Therefore, we explored whether calpain-mediated mechanisms contribute to trauma-/incubation-induced cell death.After 30 min recovery following slice preparation, we incubated the slices in aCSF containing a nonselective calpain inhibitor (MDL-28710, 30 µM) or vehicle for the duration of the experiment.We found that calpain inhibition significantly reduced the number of PI + cells across different depths (Fig. 6A,B) and resulted in faster exponential decays of PI + cells across the slice depth, even after prolonged incubation (Fig. 6C).There was also a significant decrease in the total number of PI + cells and decay constants with MDL (Fig. 6D,E).Under conditions of calpain inhibition, we also saw a significant reduction in colocalized puncta (Fig. 6F) and the number of GCaMP-filled neurons across slice depth (Fig. 6G).These findings suggest that activation of calpain-mediated processes contributes to trauma-/incubation-induced cell death in the neocortex of acute neonatal brain slices.

Discussion
We explored the spatiotemporal extent of trauma injury in acute neonatal brain slices using PI and Ca 2+ imaging as markers for cytotoxicity.We observed the following: first, trauma-induced damage was more prominent on the surface of the brain slices and exhibited a depth-dependent decrease (Fig. 1).Second, a spatial correlation exists between cell death and GCaMP-filled neurons across different depths (Fig. 2).Third, prolonged incubations exacerbated the injury in deeper tissue (Fig. 3).Fourth, GCaMP-filled neurons do not participate in network activity (Fig. 4), and there are depth-dependent alterations in seizure-induced neuronal Ca 2+ responses (Fig. 5), showing that trauma-induced injury alters neuronal responses.Finally, pharmacological inhibition of calpain during incubation mitigated trauma-induced injury and cell death, even during prolonged incubation (Fig. 6).Our results point to a calpain-mediated process downstream of Ca 2+ entry as a contributing factor for trauma-induced cell death and injury, rendering some neurons nonparticipants in network activity.
This study has certain limitations.First, our scope was limited to neonatal brain slices (P8-12).We did not expand our research to adult slices to determine if cell injury has a similar spatiotemporal evolution as neonatal ones.Second, while recent studies suggest that the viability of acute brain slices can be improved with modified cutting and incubation buffers (Kirov et al., 2004;Hájos and Mody, 2009;Ting et al., 2018;Djurisic, 2020;Perumal and Sah, 2022) or by constant aCSF recirculation through a UV filtration system (Buskila et al., 2014), we did not systematically evaluate this, as this was beyond the scope of this study.Studying every variable is onerous in time and resources.Instead, researchers may want to determine the extent of trauma-induced injury and viability of slices prepared using their protocols, at least with straightforward PI imaging, or take notice of the depth in a slice where the analysis is being conducted to increase rigor and reproducibility.Third, we did not evaluate the molecular signature of the PI + cells without GCaMP6s expression to determine their lineage.This identification was outside our goal, and future studies exploring molecular signatures of these PI + cells, either in fixed tissue or live brain slices, may address their identity.Finally, the imaging resolution was limited for detecting fluorescent signals within the somatic compartments.It was not intended to resolve physiological or morphological features of neurites, as the aim of our study was not to evaluate dendritic compartments.Future studies will address the spatiotemporal extent of neurite injury in acute slices.
Since their inception in the 1960s, acute brain slice preparations remain a critical and popular model in neuroscience research (Yamamoto and McIlwain, 1966;Dingledine et al., 1980;Collingridge, 1995).Acute brain slices are also used to model neurological disorders like epilepsy, stroke, and ischemic and traumatic brain injury (Jarvis et al., 2001;Aiba and Shuttleworth, 2012;Dzhala et al., 2012;Juzekaeva et al., 2020;Suryavanshi et al., 2022;Takezawa et al., 2023).Significantly, traumatic injury during slice preparation can induce long-lasting alterations in neuronal morphology, synaptic activity, and chloride concentrations (Reid et al., 1988;Kirov et al., 1999;Davies et al., 2007;Dzhala et al., 2012;Glykys and Staley, 2016;Glykys et al., 2017).Although most cytotoxic injury is localized to the surface of acute brain slices, recording depths are not commonly described when slices are used for physiological recordings.Also, inherent trauma-induced injury in acute brain slices can significantly confound measurements of neuronal activity caused by perturbations that model neurological disorders.Thus, experiments exploring morphological anomalies or synaptic physiology in acute brain slices are especially susceptible to inconsistent or inaccurate results if the inherent traumatic injury during the preparation of brain slices is not accounted for.Therefore, knowing the spatiotemporal extent of traumatic injury is critical when using acute brain slices.
Massive tissue trauma during acute slice preparation mimics severe traumatic brain injury (penetrating injury), resulting in cell death.Trauma in acute brain slices, besides causing physical injury, can also result in neuronal hyperexcitability and elevation in intracellular Ca 2+ (Sun et al., 2008;Szydlowska and Tymianski, 2010).Persistently elevated neuronal Ca 2+ leads to calpain activation and a cascade of downstream processes, including degradation of nuclear pore complexes and membranes during cell death (Sugiyama et al., 2017;Yamashita et al., 2017) and the enlargement of the nuclear pore complex, leading to the translocation of cytosolic GCaMP into the nucleus (Suryavanshi et al., 2024).We showed that trauma to neonatal brain slices increased the number of PI + cells and GCaMP-filled neurons, caused in part by a calpain-mediated process, as we observed that inhibiting calpains during incubation mitigated cell death and neuronal Ca 2+ overload in acute brain slices, even after prolonged incubations.Similar findings were observed when using a specific calpain-2 pharmacological inhibitor, suggesting that calpain-2 mediates most trauma-/incubation-induced cell death in acute brain slices.However, as calpain inhibition only partially prevented cell death during slice incubation, our results suggest that other calpain-independent mechanism also mediates cell death during slice trauma.These other mechanisms may include apoptosis, necrosis, necroptosis, and/or ferroptosis, as these have also been implicated in traumatic brain injury (Engel et al., 2011;Bragin et al., 2012;Piao et al., 2012;Vanden Berghe et al., 2014;Liu et al., 2016;Fricker et al., 2018).Hence, based on our results, inhibiting calpain activity may be a potential strategy to improve cellular viability after acute brain trauma, especially during the initial acute clinical presentation (Brorson et al., 1995;Rami et al., 2000;Bevers et al., 2009;Y. Wang et al., 2018).
Finally, we demonstrate that GCaMP6-filled neurons do not participate in network activity and that seizure-related Ca 2+ dynamics are altered within neocortical circuits in regions with many filled neurons."Injured" superficial tissue exhibited less frequent and less prominent seizure-related Ca 2+ spikes than "healthier" deeper tissue in the same slice.These findings indicate that a high number of injured neurons can alter network activity and disease-relevant physiological measurements.
In conclusion, we demonstrate spatiotemporal dynamics of cell injury in acute neonatal brain slices using PI labeling and neuronal Ca 2+ load as markers for cytotoxicity.We show that trauma leads to cell death (PI + ), increased GCaMP-filled neurons, and elevated neuropil Ca 2+ , which predominate in the superficial areas of the brain slices.These GCaMP-filled neurons do not participate in network activity and alter seizure dynamics.Blocking calpains decreased cellular death and the number of GCaMP-filled neurons, providing mechanistic insight and a potential strategy to prevent cell death after penetrating brain trauma.

Movie 2 .Figure 6 .
Figure 5. Depth-dependent alterations in seizure-induced neuronal Ca 2+ transients.A, Schematic depicting experimental design.B, Representative traces showing synchronous, whole-field Ca 2+ transients obtained at different depths.See Movie 3. Synchronous neuronal Ca 2+ activity in deeper tissue showed a significant increase in (C) Ca 2+ transient frequency, (D) mean amplitude, and (E) mean area under the curve (AUC), without a significant change in the Ca 2+ transient duration measured as full-width at half-maximum (F, FWHM).Wilcoxon matched pairs test, n = 6 slices.

Table 1 .
Statistical table