Abstract
GABAergic Inhibition Shapes Interictal Dynamics in Awake Epileptic Mice
Muldoon SF, Villette V, Tressard T, Malvache A, Reichinnek S, Bartolomei F, Cossart R. Brain 2015;138:2875–2890. doi:10.1093/brain/awv227.
Epilepsy is characterized by recurrent seizures and brief, synchronous bursts called interictal spikes that are present in between seizures and observed as transient events in EEG signals. While GABAergic transmission is known to play an important role in shaping healthy brain activity, the role of inhibition in these pathological epileptic dynamics remains unclear. Examining the microcircuits that participate in interictal spikes is thus an important first step towards addressing this issue, as the function of these transient synchronizations in either promoting or prohibiting seizures is currently under debate. To identify the microcircuits recruited in spontaneous interictal spikes in the absence of any proconvulsive drug or anesthetic agent, we combine a chronic model of epilepsy with in vivo two-photon calcium imaging and multiunit extracellular recordings to map cellular recruitment within large populations of CA1 neurons in mice free to run on a self-paced treadmill. We show that GABAergic neurons, as opposed to their glutamatergic counterparts, are preferentially recruited during spontaneous interictal activity in the CA1 region of the epileptic mouse hippocampus. Although the specific cellular dynamics of interictal spikes are found to be highly variable, they are consistently associated with the activation of GABAergic neurons, resulting in a perisomatic inhibitory restraint that reduces neuronal spiking in the principal cell layer. Given the role of GABAergic neurons in shaping brain activity during normal cognitive function, their aberrant unbalanced recruitment during these transient events could have important downstream effects with clinical implications.
Commentary
Interictal spikes (IIS) are transient electrographic events that, by definition, occur between spontaneous seizures in epileptic patients and animals. However, whether IIS impede or exacerbate epilepsy and their role in seizure initiation is controversial (1, 2). Although long term monitoring of electrical activity in experimental epilepsy has revealed the emergence of interictal events prior to the onset of spontaneous, epileptic seizures (1), IIS fail to show consistent spatial and temporal relationships with seizure onset (3). IIS are associated with impaired memory and cognitive function in seizure-free patients, and studies in epilepsy models suggest that they contribute to epilepsy comorbidities. Because of their occurrence as a prelude to epilepsy and correlation with memory deficits IIS are considered biomarkers for epilepsy, disease modifying, and a potential target for treatment.
IIS represent synchronous neuronal activation in the underlying cortex and are often simultaneously recorded from multiple cortical electrodes. Both synaptic inhibition and synchronous neuronal bursting have been proposed to mediate interictal discharges. Recently, studies using acute models of epileptiform activity in vitro have suggested that IIS are associated with a collapse in perisomatic inhibition resulting in enhanced pyramidal cell firing (4). However, the structure and activity in epileptogenic circuits can differ from that of normal networks, and the nature of the neuronal activity underlying IIS in epileptic circuits has remained elusive. Identifying the microcircuit activity that underlies IIS is crucial to evaluating whether these events limit or promote epileptogenesis.
The study by Muldoon et al. (2015) is a valiant step towards characterizing the hippocampal neuronal ensembles activated during IIS in chronically epileptic, pilocarpine-treated mice. By examining unanesthetized awake, behaving animals during self-paced running, the study design eliminates major confounds associated with neuronal activity changes during anesthesia and forced behaviors. They adopt a combination of electrophysiology and two-photon imaging of hippocampal CA1, simultaneously with contralateral EEG recordings, to obtain high-resolution recordings and images of neuronal populations activated during IIS. First, they use linear silicon probe recordings and kernel current source density analysis to demonstrate the presence of a current source in the CA1 pyramidal cell layer immediately following a contralateral IIS, during which CA1 multi-unit activity was consistently depressed. These data indicate strong pyramidal cell inhibition during IIS. Next, using two-photon imaging of neuronal activity patterns in CA1 stratum oriens and pyramidale of awake mice transfect-ed with the calcium indicator GCaMP, they identify a global diffuse increase in hippocampal calcium transients during IIS. In control studies using juxtacellular recordings, they show that two-photon imaging of GCaMP signals allows for the detection of single-cell activity in CA1 at low firing rates. Therefore, the diffuse pyramidal cell layer activity during IIS is consistent with activation of perisomatic axons. Adopting a clever analytical tool, they obtain a snapshot or “spatial imprint” of the dynamic changes in calcium averaged over 500 ms during and after the IIS to determine the cellular activation and quantify fluorescence change associated with an IIS. Using this “fingerprint” of activity, they confirm the presence of diffuse axon fiber activation in the pyramidal cell layer following IIS, with limited recruitment of somatic firing. In contrast, stratum oriens shows a robust increase in cellular firing in addition to diffuse neuropil activation indicating that GABAergic neurons in stratum oriens are more likely to be recruited during an IIS. Like earlier studies in vitro (5), they demonstrate differential recruitment of neuronal subpopulations during each IIS. By repeating the imaging experiments in epileptic mice expressing GCaMP in GABAergic neurons, they provide compelling evidence that IIS are associated with diffuse axonal activation of perisomatic interneuron terminals in stratum pyramidale and a robust cellular activation of GABAergic interneurons in stratum oriens. Thus, spontaneous IIS in epileptic mice recruit variable subsets of GABAergic neurons in stratum oriens and are associated with synchronous inhibition of glutamatergic neurons in the pyramidal cell layer.
Finally, using hierarchical clustering algorithms, they examine whether spatial similarity in IIS “fingerprints” correlate with similar electrographic network activity defined by IIS waveform characteristics. They find that although both shared and distinct spatial patterns of calcium activity emerged during IIS, similar patterns were rarely recruited by sequential IIS. Additionally, microcircuit activation in response to individual IIS, which showed high waveform correlations, was not always similar, confirming that variable populations of GABAergic neurons are activated during each IIS. The authors report an intriguing supralinear relationship between calcium signal and contralateral IIS amplitude. Since calcium signals can show supralinear scaling with activity and appear to recruit distinct interneuronal populations, their results imply that GABAergic microcircuit recruitment may shape global epileptiform dynamics.
The study is a significant advance towards identifying the microcircuit dynamics during IIS. While pushing the boundaries of in vivo imaging, the relatively low temporal resolution of imaging (at about 8 Hz) and the averaging of images over a 500 ms period to develop the “spatial imprint” and “similarity matrix” are substantial limitations. Interictal events are transient—lasting 100 to 250 ms—and may involve early and late phases of activity arising from distinct sources, and bursting activity in pyramidal neurons may have been missed by the temporal resolution of imaging and temporal averaging. Even though unit activity during the peak of the IIS was not resolved in silicon probe recordings, the correlation between an image analysis and unit recordings immediately after the IIS is consistent with robust inhibition. Additional concerns regarding the use of contralateral EEG and removal of cortex on the side of imaging are partly allayed by corroborating data from multisite recordings.
Using cutting-edge experimental techniques complemented by elegant analysis, the study provides a visual representation of hippocampal activity patterns during IIS in awake behaving epileptic mice and shows that IIS largely recruit inhibition. These findings differ from the proposal that inhibitory collapse underlies pathological IIS and demonstrate that GABAergic microcircuits shape interictal discharges. It remains to be examined whether methodological differences or network reorganizations known to be present in epileptic circuits contribute to the distinct mechanisms for generation of IIS in acute seizure models and epileptic circuits. Additionally, since oriens interneurons include several distinct neuronal populations, the current study paves the way for cell-type specific examination of the interneuronal classes activated during IIS. Given the diffuse calcium signaling in the cell layer, the possibility needs to be examined that basket cells in the oriens are preferentially recruited during IIS. Similarly, the potential upstream source of coordinated interneuron firing is a question of great interest. Taken together, the study identified globally synchronous firing of GABAergic neurons as guilty in shaping IIS. Global recruitment of interneuron firing may explain the cognitive comorbidities associated with IIS and bring us closer to a verdict on the protective versus pathological role of these events.