Abstract
Spampanato J, Dudek FE. eNeuro 2017;4:pii ENEURO.0130-17.2017.
The death of GABAergic interneurons has long been hypothesized to contribute to acquired epilepsy. These experiments tested the hypothesis that focal interneuron lesions cause acute seizures [i.e., status epilepticus (SE)] and/or chronic epilepsy [i.e., persistent spontaneous recurrent seizures (SRSs)]. To selectively ablate interneurons, Gad2-ires-Cre mice were injected unilaterally in the CA1 area of the dorsal hippocampus with an adeno-associated virus containing the diphtheria toxin receptor (DTR). Simultaneously, an electrode, connected to a miniature telemetry device, was positioned at the injection site for chronic recordings of local field potentials (LFPs). Two weeks after virus transfection, intraperitoneal injection of DT consistently caused focal, specific, and extensive ablation of interneurons. Long-term, continuous monitoring revealed that all mice with DT-induced interneuron lesions had SRSs. Seizures lasted tens of seconds and interseizure intervals were several hours (or days); therefore, these interneuron lesions did not induce SE. The SRSs occurred 3–5 d after DT treatment, which is the estimated time required for DT-induced cell death; therefore, induction of SRSs occurred without the latent period typical of acquired epilepsy. In five of six DT-treated mice, SRSs stopped within days, suggesting that the DT-induced interneuron lesions did not usually cause epilepsy. In one mouse, however, SRSs occurred for ≥34 d after interneuron ablation, similar to epilepsy after experimental SE. Sham control mice had no detectable seizures, confirming that the SRSs were due to ablation of interneurons. These data show that selective interneuron ablation consistently caused SRSs but not SE; and, at least under the conditions used here, interneuron lesions rarely led to persistent SRSs (i.e., epilepsy).
Drexel M, Romanov RA, Wood J, Weger S, Heilbronn R, Wulff P, Tasan RO, Harkany T, Sperk G. J Neurosci 2017;37:8166–8179.
Temporal lobe epilepsy (TLE) is the most frequent form of focal epilepsies and is generally associated with malfunctioning of the hippocampal formation. Recently, a preferential loss of parvalbumin (PV) neurons has been observed in the subiculum of TLE patients and in animal models of TLE. To demonstrate a possible causative role of defunct PV neurons in the generation of TLE, we permanently inhibited GABA release selectively from PV neurons of the ventral subiculum by injecting a viral vector expressing tetanus toxin light chain in male mice. Subsequently, mice were subjected to telemetric EEG recording and video monitoring. Eighty-eight percent of the mice presented clusters of spike-wave discharges (C-SWDs; 40.0 ± 9.07/month), and 64% showed spontaneous recurrent seizures (SRSs; 5.3 ± 0.83/month). Mice injected with a control vector presented with neither C-SWDs nor SRSs. No neurodegeneration was observed due to vector injection or SRS. Interestingly, mice that presented with only C-SWDs but no SRSs, developed SRSs upon injection of a subconvulsive dose of pentylenetetrazole after 6 weeks. The initial frequency of SRSs declined by ⊠30% after 5 weeks. In contrast to permanent silencing of PV neurons, transient inhibition of GABA release from PV neurons through the designer receptor hM4Di selectively expressed in PV-containing neurons transiently reduced the seizure threshold of the mice but induced neither acute nor recurrent seizures. Our data demonstrate a critical role for perisomatic inhibition mediated by PV-containing interneurons, suggesting that their sustained silencing could be causally involved in the development of TLE. SIGNIFICANCE STATEMENT: Development of temporal lobe epilepsy (TLE) generally takes years after an initial insult during which maladaptation of hippocampal circuitries takes place. In human TLE and in animal models of TLE, parvalbumin neurons are selectively lost in the subiculum, the major output area of the hippocampus. The present experiments demonstrate that specific and sustained inhibition of GABA release from parvalbumin-expressing interneurons (mostly basket cells) in sector CA1/subiculum is sufficient to induce hyperexcitability and spontaneous recurrent seizures in mice. As in patients with nonlesional TLE, these mice developed epilepsy without signs of neurodegeneration. The experiments highlight the importance of the potent inhibitory action mediated by parvalbumin cells in the hippocampus and identify a potential mechanism in the development of TLE.
Commentary
It seems self-evident: Reduced inhibitory drive in the brain increases neuronal excitability and leads to seizures, which in turn promotes epileptogenesis. Yet, this seemingly simple relationship has not been directly proven so far. Since the first documentation of reduced GABAergic innervation at sites of seizure foci in a monkey model of epilepsy decades ago (1), loss of GABAergic interneurons has been shown in many different animal models and in humans with epilepsy. A few studies have demonstrated that ablation of subtypes of interneurons during development in mice can lead to spontaneous seizures (2). However, to date, it is still controversial if loss of interneurons is causal or consequential to epilepsy and, most importantly, if it contributes to epileptogenesis. Two elegant studies by Drexel and colleagues and Spampanato and Dudek have now begun to shed light on these questions by showing, for the first time, that focal adult-onset ablation or inactivation of GABAergic interneurons can lead to transient spontaneous recurrent seizures in mice without direct evidence for epileptogenesis (persistent epilepsy).
The two studies came to similar conclusions although they employed very different strategies to reduce GABAergic interneuron activity in the hippocampus. In a small-scale “proof-of-principle” study, Spampanato and Dudek used a diphtheria toxin-based approach (3) to selectively ablate a subset of GAD67-positive interneurons in the dorsal CA1 region. In contrast, Drexel and colleagues virally expressed tetanus toxin light chain in parvalbumin-positive interneurons of the subiculum and the CA1, which permanently inactivates synaptic transmission by blocking vesicular release of neurotransmitters but leaves the neurons alive (4). Both strategies ablated or silenced interneurons unilaterally in limited areas of the subiculum or the CA1 region, as shown convincingly by immunohistochemical and electrophysiological methods. This focal inhibition of GABAergic transmission was sufficient to induce spontaneous recurrent seizures in all animals (Spampanato and Dudek), or EEG abnormalities, namely clusters of spike–wave discharges (C-SWDs) and spontaneous recurrent seizures in most cases (Drexel et al.). The methods to detect seizure activity—measuring local field potentials paired with behavioral analyses through video recordings—were very similar in the two studies. Therefore, the variances in phenotype severity were most likely not due to differences in seizure detection sensitivity. Instead, it is conceivable that the complete ablation of roughly 88% of GAD67-positive interneurons in a focal region of the hippocampus has a more profound effect on neuronal network stability than the inactivation of about 62% of parvalbumin-positive neurons in the hippocampus, thereby leading to the more severe phenotype observed in the ablation study by Spampanato and Dudek. Drexel and colleagues’ approach to selectively inactivate parvalbumin-positive interneurons in the subiculum, which reflects observations in patients with temporal lobe epilepsy (5), led to increased susceptibility to pentylenetetrazole-induced seizures even in the absence of spontaneous seizures or C-SWDs, suggestive of an overall hyperexcitable state. Loss of inhibition had to be permanent to be effective: Transient inhibition of parvalbumin-positive interneurons in the subiculum for less than two hours using designer receptors exclusively activated by designer drugs (DREADDs) did not lead to EEG abnormalities or recurrent seizures. Notably, neither of the two studies reported induction of status epilepticus after focal loss of GABAergic transmission.
Perhaps the most surprising aspect of these studies is that neither of the two approaches provided experimental support for the hypothesis that loss of GABAergic interneuron activity induced progressive epilepsy and epileptogenesis, at least not during the time the mice were monitored (6 to 10 weeks). Indeed, spontaneous recurrent seizures ceased over time in five out of six mice when neurons were ablated (Spampanato and Dudek), or declined in frequency after 5 to 6 weeks in the synaptic inactivation study (Drexel et al.). Interneuron ablation that is too restricted, spatially or subtype-specific, or potential compensatory changes in the network may explain these observations. It is important to note that Spampanato and Dudek showed that spontaneous recurrent seizures occurred without a clear latent phase and basically started at the same time that neuronal cell death began. This finding could be interpreted as an acute seizure phase after injury; in humans, this would be followed by a sometimes years-long latent phase before the development of epilepsy. Similarly, in animal models of mild traumatic brain injury, spontaneous recurrent seizures can occur months after injury. Thus, 10 weeks of seizure recording after interneuronal loss might not have been long enough to detect enduring progressive epilepsy.
The lack of clear evidence for epileptogenesis in both studies is even more notable considering the different approaches the studies followed. In one study, interneurons were permanently silenced but remained in the network without obvious neurodegeneration, providing at least structural support. In the other study, the interneurons were effectively killed, triggering processes of phagocytosis. If compensatory changes would explain lack of persistent recurrent seizures and epileptogenesis, they would probably be very different in the two models. This makes the “two-hit model,” as discussed by Spampanato and Dudek, rather likely, positing that other mechanisms apart from loss of GABAergic inhibition (such as certain genetic susceptibilities) may be needed to induce progressive epilepsy. The hypothesis could be tested in the future by assessing if combining the reduction of interneuron activity with the genetic manipulation of epilepsy susceptibility genes is sufficient to trigger epileptogenesis.
The interplay between GABAergic signaling and seizure regulation in epilepsy is complex and involves several different brain regions. Further work is needed, in the same vein as the two studies discussed here, to provide a comprehensive picture of the role of interneuronal loss in brain regions other than the hippocampus on the development of seizures and progressive epilepsy. The influence of GABAergic inhibition on seizure susceptibility may also differ depending on the subcellular localization of GABA receptors involved: GABAergic inhibition can be phasic (mediating inhibitory postsynaptic currents through synaptic GABA receptors) or tonic (mediating a permanent current through extrasynaptic GABA receptors). In absence epilepsy, prior work suggested that an increase in tonic GABAergic inhibition in thalamocortical neurons contributed to seizure development, while phasic inhibition was unchanged (6). Tonic GABAergic inhibition has been observed in hippocampal interneurons (7), and future studies are therefore needed to assess how tonic GABAergic inhibition is affected by focal silencing or ablation of select interneurons in the hippocampus.
Recent preclinical studies demonstrated that increasing inhibition through transplanting interneurons into epileptic brains can reduce seizures in rodent epilepsy models (8). Advances in stem cell technology and the capability to generate human neurons from induced pluripotent stem cells make this a more feasible and likely potential therapeutic strategy for epilepsy in humans. The reviewed studies emphasize the need for further work to fully explore the role of loss of inhibition in triggering epileptogenesis and whether compensating for reduced inhibition will indeed be sufficient to prevent epilepsy progression.