Mild systemic inflammation and moderate hypoxia transiently alter neuronal excitability in mouse somatosensory cortex

Abstract

During the perinatal period, the brain is highly vulnerable to hypoxia and inflammation, which often cause white matter injury and long-term neuronal dysfunction such as motor and cognitive deficits or epileptic seizures. We studied the effects of moderate hypoxia (HYPO), mild systemic inflammation (INFL), or the combination of both (HYPO+INFL) in mouse somatosensory cortex induced during the first postnatal week on network activity and compared it to activity in SHAM control animals. By performing in vitro electrophysiological recordings with multi-electrode arrays from slices prepared directly after injury (P8-10), one week after injury (P13-16), or in young adults (P28-30), we investigated how the neocortical network developed following these insults. No significant difference was observed between the four groups in an extracellular solution close to physiological conditions. In extracellular 8mM potassium solution, slices from the HYPO, INFL, and HYPO+INFL group were more excitable than SHAM at P8-10 and P13-16. In these two age groups, the number and frequency of spontaneous epileptiform events were significantly increased compared to SHAM. The frequency of epileptiform events was significantly reduced by the NMDA antagonist D-APV in HYPO, INFL, and HYPO+INFL, but not in SHAM, indicating a contribution of NMDA receptors to this pathophysiological activity. In addition, the AMPA/kainate receptor antagonist CNQX suppressed the remaining epileptiform activity. Electrical stimulation evoked prominent epileptiform activity in slices from HYPO, INFL and HYPO+INFL animals. Stimulation threshold to elicit epileptiform events was lower in these groups than in SHAM. Evoked events spread over larger areas and lasted longer in treated animals than in SHAM. In addition, the evoked epileptiform activity was reduced in the older (P28-30) group indicating that cortical dysfunction induced by hypoxia and inflammation was transient and compensated during early development.

Keywords: Barrel cortex; Development; Electrophysiology; Epileptiform activity; Hypoxia; In vitro; Interleukin-1β; Mouse; Multi-electrode array; Systemic inflammation.

Figure 1. Hypoxia and/or systemic inflammation increase the spontaneous firing rate

A) Representative picture of a thalamocortical slice on a MEA prepared from a mouse of the P8–10 age group. B) Spike sorting of individual action potentials recorded over a 10 minute period from the slice in (A) on each of the 60 electrodes show low activity present in all cortical layers and in the hippocampus. Some electrodes could record the activity of more than one neuron. C), D), and E) Evolution of the MUA (in Hz) for neurons located in cortical layers 2 to 6 during 5 minutes recordings in ACSF with 8 mM KCl in representative slices from the P8–10 group (C), the P13–16 group (D), and the P28–30 group (E). For each condition, a raster plot shows the MUA on all MEA electrodes located in all layers of the Barrel cortex (bottom) with the total spike frequency of all these channels (bin: 1 sec, top). Note the peaks of activity corresponding to epileptiform events. Spikes occurring during these events were not counted in the spontaneous firing rate. F) Average firing rate per layers of the Barrel cortex recorded in thalamocortical slices from mice of P8–10 (left), 13–16 (middle) and P28–30 age groups (right) treated with all four conditions: SHAM (dark grey), HYPO (blue), INFL (red) and HYPO+INFL (green). G) The mean cortical firing rate in a ten minute recording period per slice and condition is plotted as box and whisker plots. For each age group, the mean firing rate is plotted for 3 mM (open boxes) and 8 mM (filled boxes) KCl ACSF. The numbers of slices recorded per condition are represented on the top of the graph.

Figure 2. Hypoxia and/or systemic inflammation significantly increase spontaneous epileptiform activity in slices from P8–10 and P13–16 animals

A), B), and C) Representative MEA recordings of spontaneous epileptiform events occurring in the Barrel cortex of thalamocortical slices perfused with ACSF containing 8 mM KCl from a P8–10 mouse (A), P13–16 mouse (B), and P28–30 mouse (C) for all four treatment conditions. For each condition, a 30 s recording on all 60 electrodes is showed with epileptiform events occurring (top), the trace on one electrode marked by a dark gray square is magnified (middle), and one single epileptiform recorded from this electrode marked in light gray is further magnified (bottom). D) The mean frequencies of epileptiform events occurrence in a ten minute recording period per slice and condition is plotted as box and whisker plots. For each age group, the mean number of occurrence is plotted for 3 mM (open boxes) and 8 mM (filled boxes) KCl-ACSF for SHAM (dark grey), HYPO (blue), INFL (red) and HYPO+INFL (green). Dunns test: **, p<0.01; *, p<0.05.

Figure 3. Pharmacological properties of epileptiform events induced by hypoxia and inflammation

A) Examples of EEs recorded from a slice prepared from a P13–16 HYPO animal with different recording conditions. For each of the recording solutions indicated, 500 ms recording traces show the activity recorded by each of the 60 electrodes of the MEA during a spontaneously occurring EE. The recording trace on the electrode marked by a grey square is magnified below. APV decreases the number of events compared to 8 mM KCl-ACSF alone while addition of CNQX suppresses it. B) The mean frequencies of EEs occurrence in a ten minute recording period per slice and per condition is plotted as box and whisker plots (SHAM, dark grey; HYPO, blue; INFL, red; HYPO+INFL, green). For each age group, the mean number of occurrence is plotted for 8 mM KCl ACSF (open boxes), APV alone (light filled boxes) or APV and CNQX together (dark filled boxes). Dunns test: **, p<0.01; *, p<0.05.

Figure 4. Bicuculline increase EEs frequency and amplitude in all conditions

Examples of EEs recorded from a slice prepared from a P8–10 HYPO animal in ACSF containing 8 mM KCl alone or together with bicuculline. For both recording solutions, 500 ms recording traces show the activity recorded by each of the 60 electrodes of the MEA during a spontaneous EE. The recording trace on the electrode marked by a grey square is magnified below. Note the larger spreading of the events with bicuculline, as well as the longer duration and higher amplitude. B) The mean frequencies of EEs occurrence in a ten minute recording period per slice and per condition is plotted as box and whisker plots (SHAM, dark grey; HYPO, blue; INFL, red; HYPO+INFL, green). For each age group, the mean number of occurrence is plotted for 8 mM KCl ACSF (open boxes), and 8 mM KCL-ACSF + Bic (filled boxes). C) Increase of the amplitude, D) duration, and E) number of electrodes with synchronous activity of epileptiform events in presence of Bic for each condition. Dunns test: **, p<0.01; *, p<0.05.

Figure 5. Evoked field response is not altered by hypoxia and/or systemic inflammation

A) Example traces of the evoked field potentials recorded by 5 electrodes in the layers 2/3 and 4 surrounding the electrically stimulated electrode in layer 4 (2 V, channel without recording trace) for each age and treatment condition (SHAM, dark grey; HYPO, blue; INFL, red; HYPO+INFL, green) in ACSF with 3 mM KCl. The field potentials marked by a colored square, corresponding to the layer 2/3 of the stimulated column, were used to calculate the input-output curves. B) Input-output curves plotted for all four treatments and all three age groups. Slices were stimulated with a monopolar biphasic stimulus in layer 4 varying between 250 mV and 4 V. The half maximum voltage was similar for all condition in each age group. C) Amplitude of the evoked field potentials for each age group and each condition in 3 mM KCl-ACSF (open boxes), 8 mM KCl-ACSF (light filled boxes) and 8 mM KCl-ACSF + 50 μM APV (dark filled boxes). Dunns test: **, p<0.01; *, p<0.05.

Figure 6. Synaptic activity between L4 and L2/3 is not altered by hypoxia and/or systemic inflammation

A) Example traces of the evoked responses in L2/3 to paired-pulse stimulation in L4 (2 V) with 25 ms between both stimuli for each age and treatment condition (SHAM, dark grey; HYPO, blue; INFL, red; HYPO+INFL, green) recorded in ACSF with 3 mM KCl. B) Paired-pulse facilitation measured for each treatment condition in P13–16 mice in ACSF with 3 mM KCl. The graphs represent the average PPR obtained from all slices of the different ages and treatments at varying interstimulus intervals. C) Example traces of the first and last evoked responses in L2/3 to 100 pulses at 10 Hz in L4, recorded in ACSF with 8 mM KCl. D) Synaptic fatigue recorded for 100 stimuli at 10 Hz (2 V, 8 mM KCl-ACSF). The graphs represent the relative amplitude of the evoked responses in slices prepared from all treated animals relative to the response to the first stimulus.

Figure 7. Increase neuronal excitability in hypoxia and/or IL-1β treated animals

A) Representative 500 ms recordings of the evoked responses to monopolar stimulations in the layer 4 at 2 V for each treatment in slices prepared from P8–10 mice (top), P13–16 (middle), and P28–30 mice (top). The position of the cortical layers is indicated. The electrodes used for stimulation and calculation of evoked EEs durations are located above the stimulation electrode, marked by the absence of recording, as it was switched off during voltage application. B) Average minimum stimulation voltage needed to initiate an epileptiform response in each treatment (SHAM, dark grey; HYPO, blue; INFL, red; HYPO+INFL, green) in 3 mM KCl- (open bars) or 8 mM KCl-ACSF (filled bars). C) Duration of the evoked responses recorded in layer 2/3 following a 2.5 V stimulation in layer 4 presented as box-and-whisker plots representing the median as a solid line with a box to indicate the interquartile range; the whiskers represent the 10th and 90th percentiles. D) Similar box and whiskers plots representing the median number of barrel columns over which the evoked response was propagating following stimulation in layer 4. Dunns test: **, p<0.01; *, p<0.05.