Neuronal Circuit Activity during Neonatal Hypoxic-Ischemic Seizures in Mice

Abstract

Objective: To identify circuits active during neonatal hypoxic-ischemic (HI) seizures and seizure propagation using electroencephalography (EEG), behavior, and whole-brain neuronal activity mapping.

Methods: Mice were exposed to HI on postnatal day 10 using unilateral carotid ligation and global hypoxia. EEG and video were recorded for the duration of the experiment. Using immediate early gene reporter mice, active cells expressing cfos were permanently tagged with reporter protein tdTomato during a 90-minute window. After 1 week, allowing maximal expression of the reporter protein, whole brains were processed, lipid cleared, and imaged with confocal microscopy. Whole-brain reconstruction and analysis of active neurons (colocalized tdTomato/NeuN) were performed.

Results: HI resulted in seizure behaviors that were bilateral or unilateral tonic-clonic and nonconvulsive in this model. Mice exhibited characteristic EEG background patterns such as burst suppression and suppression. Neuronal activity mapping revealed bilateral motor cortex and unilateral, ischemic somatosensory cortex, lateral thalamus, and hippocampal circuit activation. Immunohistochemical analysis revealed regional differences in myelination, which coincide with these activity patterns. Astrocytes and blood vessel endothelial cells also expressed cfos during HI.

Interpretation: Using a combination of EEG, seizure semiology analysis, and whole-brain neuronal activity mapping, we suggest that this rodent model of neonatal HI results in EEG patterns similar to those observed in human neonates. Activation patterns revealed in this study help explain complex seizure behaviors and EEG patterns observed in neonatal HI injury. This pattern may be, in part, secondary to regional differences in development in the neonatal brain. ANN NEUROL 2019;86:927-938.

FIGURE 1:

Seizure characteristics in p10 mice exposed to neonatal hypoxia–ischemia. (A) Representative power spectrogram from the ischemic parietal cortex electrode through the experimental timeline. Timed injection of 4-hydroxytamoxifen (4OHT; syringe icon), 30 minutes after the end of hypoxia, to capture a 90-minute window of neuronal activity (pink box; amplitude color heat map scale ×10^–6). Arrows indicate the time that raw electroencephalogram tracings below the spectrogram represent. (B) Seizure behaviors for the entire experiment, postischemia/prehypoxia, during hypoxia, and posthypoxia. (C) Behavioral seizure score (BSS) and timing for all seizure events (n = 30 mice, each mouse has a unique symbol, each point is a discrete seizure event). One hundred percent of mice seized during hypoxia (blue box; time = −60 minutes is the completion of carotid ligation, time = 0 is the start of hypoxia). Thirteen percent died during hypoxia following a convulsive seizure (grade 5–6). TRAP = targeted recombination in active populations.

FIGURE 2:

Characteristic electroencephalography (EEG) patterns during hypoxia ischemia. (A) EEG background from left to right: preinjury baseline, burst suppression during hypoxia, posthypoxia suppression. Recording from ipsilateral parietal cortex depth electrode. (B) Evolution of a seizure during hypoxia. Recording from ipsilateral hippocampal depth electrode.

FIGURE 3:

Increased neuronal activity in ischemic hemisphere of hypoxic–ischemic (HI) mice. (A) Representative transverse slices (200μm-thick clarified tissue, 10× tiled image from bregma −2.96) from female HI (top), male sham (middle), and male hypoxia (H) only (bottom) with active cells, tagged with tdTomato, and neurons expressing NeuN (green). Hemisphere ipsilateral (IL) to carotid ligation/ischemia or neck incision in sham group on the left in all slices, contralateral (CL) hemisphere on the right. (B) Quantification of active neurons (percent of cells both TRAPed and expressing NeuN) compared by hemisphere. Ischemic hemisphere had significantly more active neurons compared to the HI CL hemisphere (p = 0.00001) and sham IL hemisphere (p = 0.00001). The HI CL hemisphere contained more active neurons than the sham IL (p = 0.013). Sham mice did not exhibit any significant difference between hemispheres (p = 0.832). *p < 0.05, **p < 0.001 (n = 5 [2 male/3 female] HI, n = 5 [3 male/2 female] sham). (C) Asymmetric power spectrogram in HI mice during hypoxia (45-minute period) in ischemic cortex (left) and contralateral cortex (right; amplitude scale ×10^–6). Burst suppression pattern and seizures in ischemic hemisphere, suppression in CL hemisphere. (D) Background suppression during hypoxia and reoxygenation in IL and CL hemispheres. All measurements of mean voltage taken from 10-second random excerpts of the encephalogram over the experimental time period (baseline, 30 minutes postligation, during hypoxia—15 minutes and 30 minutes after start, after reoxygenation—15 minutes and 60 minutes after start) were compared to baseline. Each animal’s baseline served as its own control, and data are reported as a percentage of baseline (n = 5 mice). Measurements were taken from cortical electrodes.

FIGURE 4:

Differential asymmetries in neuronal activity in the somatosensory cortex and motor cortex of hypoxic–ischemic (HI) mice. (A) Asymmetric, increased neuronal activity in ischemic somatosensory cortex of a female HI mouse (10× tiled slice at bregma −1.23). Dashed boxes represent area pictured in C. (B) Symmetric neuronal activity in motor cortex (10× tiled slice at bregma 1.69). Dashed boxes represent area pictured in D. (C) Neuronal activity in ipsilateral (IL) and contralateral (CL) somatosensory cortex, most prominent in layers II/III and V (20× tiled slice at bregma −1.23). (D) Neuronal activity in IL and CL motor cortex, most prominent in layers II/III (20× tiled slice at bregma 1.69). (E) More tdTomato containing axons crossing midline in the anterior corpus callosum (right) than posterior callosum (left; 20× magnification). (F) Myelin basic protein (MBP) expression in anterior corpus callosum (right) and minimal expression in the posterior corpus callosum (left; 20× magnification).

FIGURE 5:

Increased neuronal activity in the ischemic lateral thalamus. (A) Active neurons in the lateral thalamic nuclei including ventroposterolateral (VPL), ventroposteriomedial (VPM), and posteriomedial (Po; 20× magnification, left and 10× magnification tiled slice, right, at bregma −1.91, female HI mouse). (B) Symmetric activation in the midline thalamic nuclei. Paraventricular (PV), centromedian (CM), and intermediodorsal (IMD) nuclei. Boxed inset in A and B with neurons colocalizing tdTomato and NeuN. CL = contralateral; IL = ipsilateral.

FIGURE 6:

Astrocytes and blood vessel endothelial cells expressing cfos in hypoxic–ischemic mice. (A) tdTomato-tagged active cells colocalized with astrocytic marker, glial fibrillary acidic protein in hippocampus (20× magnification, tiled). (B) tdTomato tagged active cells lining blood vessels, colocalized with endothelial cell marker CD31 antibody (20× magnification, tiled). High resolution insets indicated by the white box outline in merged images.

FIGURE 7:

Neuronal activity in ischemic hippocampal-parahippocampal circuit. (A) Active neurons (colocalized tdTomato and NeuN) in dentate granule (DG) cells and CA1 in coronal slice (20× magnification). (B) Transverse section through hippocampal regions displaying active neurons in CA2, CA3, and DG. (C) Active neurons in a transverse section of medial entorhinal cortex (CEnt) and parasubiculum (PaS). Perirhinal cortex (PRh) and presubiculum (PrS) also display active neurons. Boxed inset in A, B, and C with neurons colocalizing tdTomato and NeuN.

FIGURE 8:

Comparison of targeted recombination in active populations (TRAP) technique and cfos immunohistochemistry. (A) TRAPed tdTomato activity (tagging cells expressing cfos) in motor cortex following hypoxic–ischemic (HI), 10× magnification, NeuN expression (green channel). (B) Immunohistochemistry for cfos and neuN in motor cortex from tissue 2 hours following HI.