Direct Imaging of Hippocampal Epileptiform Calcium Motifs Following Kainic Acid Administration in Freely Behaving Mice

Abstract

Prolonged exposure to abnormally high calcium concentrations is thought to be a core mechanism underlying hippocampal damage in epileptic patients; however, no prior study has characterized calcium activity during seizures in the live, intact hippocampus. We have directly investigated this possibility by combining whole-brain electroencephalographic (EEG) measurements with microendoscopic calcium imaging of pyramidal cells in the CA1 hippocampal region of freely behaving mice treated with the pro-convulsant kainic acid (KA). We observed that KA administration led to systematic patterns of epileptiform calcium activity: a series of large-scale, intensifying flashes of increased calcium fluorescence concurrent with a cluster of low-amplitude EEG waveforms. This was accompanied by a steady increase in cellular calcium levels (>5 fold increase relative to the baseline), followed by an intense spreading calcium wave characterized by a 218% increase in global mean intensity of calcium fluorescence (n = 8, range [114-349%], p < 10(-4); t-test). The wave had no consistent EEG phenotype and occurred before the onset of motor convulsions. Similar changes in calcium activity were also observed in animals treated with 2 different proconvulsant agents, N-methyl-D-aspartate (NMDA) and pentylenetetrazol (PTZ), suggesting the measured changes in calcium dynamics are a signature of seizure activity rather than a KA-specific pathology. Additionally, despite reducing the behavioral severity of KA-induced seizures, the anticonvulsant drug valproate (VA, 300 mg/kg) did not modify the observed abnormalities in calcium dynamics. These results confirm the presence of pathological calcium activity preceding convulsive motor seizures and support calcium as a candidate signaling molecule in a pathway connecting seizures to subsequent cellular damage. Integrating in vivo calcium imaging with traditional assessment of seizures could potentially increase translatability of pharmacological intervention, leading to novel drug screening paradigms and therapeutics designed to target and abolish abnormal patterns of both electrical and calcium excitation.

Keywords: calcium imaging; electroencephalography; freely behaving mice; kainic acid; seizure.

Figure 1

Position of the implanted EEG electrodes and the microendoscopic calcium imaging device for the combined telemetric and calcium imaging recordings. (A) Schematic representation of the imaging device and the EEG electrodes. (B) Optical guide tube (green circle) stereotaxically centered over AP = −2.3mm, ML = 1.89 mm, V = −1.6mm from Bregma. The EEG elecrodes (blue circles) for the whole-brain EEG were implanted in the frontal cortex and superior/inferior colliculus, respectively.

Figure 2

Overview of Epileptiform Calcium Motifs following KA Administration (15 mg/kg s.c.) in Hippocampal CA1 of a Freely Behaving Mouse. (A) Simultaneous EEG (green) and calcium (black) activity following KA administration (arrow). The animal in this example never reached a convulsive motor seizure (CMS). Background shading indicates EEG seizure scores. The three stereotypical events are indicated: rapid-flashing and build-up (B), calcium waves (C), and slow-flashing (D). (B–D) Expanded views illustrating patterns of rapid flashing and cellular calcium build-up (B), calcium wave (C), and slow flashing (D). The imaging frames (top) show the levels of fluorescence at different time points (Scale: 20 μm, horizontal bar; and 0–200% change in fluorescence, vertical gray scale bar). The mean frame intensity (black) and EEG (green) traces are plotted at the bottom with the imaging frame positions indicated by the numbers 1–6.

Figure 3

Analysis of Cellular Events in Hippocampal CA1 of the Mice Treated with KA. (A) Five representative cellular calcium traces prior to KA administration; detected calcium transient events indicated by “x” marks. (B) Same traces after KA administration but before calcium build-up. (C) Same traces during the calcium build-up; same scale as in (A,B). (D) The peaks (red x) and integrals (cyan shading) of a representative cell: the 5 largest post-vehicle events (top) compared to a typical build-up event (bottom, same scale as on top). The events' beginning and end are marked with blue “x”s. (E) Distributions of the build-up event peak amplitude (red histogram, top) and integral (cyan histogram, bottom) combined across animals (₁₀log x-axis scale).

Figure 4

Epileptiform motifs in CA1 of Mice Pre-Treated with VA (300 mg/kg, i.p.) 15 min Prior to KA Administration. (A–D) A representative session (A) of an animal pre-treated with VA before KA administration (VAKA) exhibited isolated flashing (B), but otherwise had similar epileptifom patterns (C,D) as animals treated with KA only. (E) Occurrences of stereotypical events (Δ, rapid-flashing and build-up; ^*, calcium wave; __, slow-flashing) are marked in four representative VAKA (top) and KA (bottom) sessions. Background color indicates EEG seizure scores. Red “x” indicates the onset of CMS. (F) Mean normalized fluorescence intensities during the first identifiable wave were aligned with respect to peak time and plotted across time (left: KA animals; right: VAKA animals). (G) Analysis of individual neurons with respect to the characteristics of calcium-build-up events (the Log Peak Ratio and Log Integral Ratio) showed no significant differences in the VAKA group compared with the KA group.