Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy (SMEI), is a devastating drug-resistant epileptic encephalopathy, characterized by febrile/hyperthermia seizures at onset and later development of afebrile seizures, cognitive impairment, elevated mortality, and ataxia (Dravet et al., 2005). About 80% of patients with DS carry heterozygous missense or truncating mutations in the SCN1A gene encoding the NaV1.1 sodium channel α subunit (Claes et al., 2001; Marini et al., 2011), which cause haploinsufficiency: a 50% reduction of functional channels without negative dominance (Bechi et al., 2012). Notably, NaV1.1 is the target of hundreds of epileptogenic mutations causing different types of epilepsy (Catterall et al., 2010; Mantegazza et al., 2010; Marini & Mantegazza, 2010).
Heterozygous NaV1.1 knockout mice (NaV1.1+/−), in which deletion of exon 26 in Scn1a leads to haploinsufficiency of NaV1.1, recapitulate the phenotype of DS: they exhibit hyperthermia-induced and spontaneous seizures, premature death, mild ataxia, and cognitive impairment (Yu et al., 2006; Kalume et al., 2007; Oakley et al., 2009; Han et al., 2012). Although NaV1.1 starts to be expressed in rodents at about postnatal day 10 (P10; Beckh et al., 1989; Ogiwara et al., 2007), NaV1.1+/− mice show hyperthermia-induced seizures after P19 and spontaneous seizures after around P30 (Oakley et al., 2009). A knock-in mouse model of the R1407X NaV1.1 truncating mutation shows a similar phenotype (Ogiwara et al., 2007; Ito et al., 2012). These features are consistent with the patients' phenotype (Dravet et al., 2005) and set the beginning of the epileptic period in NaV1.1+/− mice at about P19, identifying a preepileptic period in which the underlying cause of epilepsy (NaV1.1 mutation) is present but there are no seizures. Electrophysiologic studies in dissociated hippocampal neurons from DS mice have shown selective loss of sodium current and excitability in γ-aminobutyric acid (GABA)ergic interneurons (Yu et al., 2006), and some immunohistochemistry studies have shown selective expression of NaV1.1 in GABAergic neurons (Ogiwara et al., 2007). These results point to selective impairment of GABAergic interneurons in DS mice, a hypothesis that has been confirmed by recent studies (Cheah et al., 2012; Dutton et al., 2012). Recently, it has also been shown that GABAergic inhibition is actually reduced in brain slices from DS mice (Han et al., 2012).
However, little is known on the properties of network hyperexcitability and on the mechanism of seizure generation in these models. In fact, properties of local circuits have not been investigated and seizures have been studied thus far with surface electroencephalography (EEG) (Yu et al., 2006; Ogiwara et al., 2007; Oakley et al., 2009), which did not show if specific brain regions are particularly involved. Notably, some studies have revealed, in comparison with the hippocampus, lesser impairments of NaV1.1 haploinsufficiency on GABAergic neuron functions in the cerebellum, reticular nucleus of the thalamus, and cerebral cortex (Yu et al., 2006; Kalume et al., 2007; Abe et al., 2010; Kalume et al., 2007), although there are limited studies done in brain slices. Shedding light on these issues is important for identifying specific network activities and brain regions that may be particularly implicated, which may be used as targets in drug screenings for the development of therapeutic approaches.
We have performed electrophysiologic recordings in brain slices from NaV1.1+/− mice in the period preceding the appearance of behavioral seizures (preepileptic period: P14–P18) and in the epileptic period (P30–P36), and intracerebral recordings with depth electrodes, identifying specific epileptic activities and network hyperexcitability generated in the hippocampus.
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We have identified signs of network hyperexcitability, epileptiform activities, and modifications of network properties that characterize neuronal circuits in a mouse model of DS, and shown that the hippocampus is particularly involved in seizure generation.
With our experimental system in vitro, we did not observe spontaneous epileptiform activities in control conditions and hyperthermia-induced spreading depression both in NaV1.1+/− and NaV1.1WT slices. Therefore, to generate epileptiform discharges and evaluate their properties in NaV1.1+/− slices, we used the convulsant 4-AP, a common inducer of epileptiform activity in vitro (Avoli & de Curtis, 2011). Application of 4AP was able to induce epileptiform activity both in NaV1.1WT and NaV1.1+/−. In NaV1.1WT, we observed the standard activity reported in rodent slices upon application of 4-AP, with ictal-like activity and interictal like-activities observed both in combined entorhinal cortex-hippocampus horizontal slices and in coronal slices comprising the somatosensory/visual cortex and the dorsal hippocampus. In slices from NaV1.1+/− mice, we observed a specific epileptiform activity that we named recurrent multicomponent discharges (RMDs) and that was present only during the epileptic period (P30–36). When RMDs were present, we did not observe standard 4-AP ictal activities generated by the entorhinal cortex in horizontal slices (in which connections between the hippocampus and the entorhinal cortex are preserved). Notably, in coronal slices (in which there are no connections between the hippocampus and the cortex) RMDs were generated in the hippocampus, whereas the somatosensory cortex generated standard 4-AP ictal-like activities, suggesting that the hippocampus proper is sufficient for their generation, that hippocampocortical connections are necessary for inhibiting the generation of standard 4-AP ictal-like activities, and that the 4-AP–induced activity in the somatosensory/visual cortex is not modified by NaV1.1 haploinsufficiency. We observed long-lasting (up to tens of seconds) RMDs only in horizontal slices, suggesting that entorhinal cortex and parahippocampal structures are important for sustaining these discharges or that the ventral hippocampus is more prone to the their generation.
RMDs are inhibited by the block of glutamatergic receptors, showing that excitatory neurotransmission is essential for their generation, similarly to preictal activities observed in human hippocampus resected from temporal lobe epilepsy patients (Huberfeld et al., 2011). In these conditions, we observed GABAergic field potentials that were similar to those that have been proposed to be important for inducing standard 4-AP ictal-like discharges in the entorhinal cortex (Barbarosie et al., 2002; Avoli & de Curtis, 2011). Notably, we found that the frequency of these GABAergic potentials is reduced in NaV1.1+/− slices from the epileptic period, whereas in the preepileptic period we found just a trend toward a reduction. This is further evidence that GABAergic activity is reduced in NaV1.1+/− neuronal networks (Yu et al., 2006; Ogiwara et al., 2007; Han et al., 2012), but with a more prominent reduction in the epileptic period, and that standard 4-AP ictal-like discharges may not be a specific ictal-like activity of NaV1.1+/− slices.
However, are RMDs specific epileptic activities of NaV1.1+/− slices? It has been reported that it is difficult to induce typical 4-AP ictal-like activities in slices of epileptic tissue obtained from human specimens or animal models of epilepsy, and this has been interpreted as a homeostatic modification of the network (Zahn et al., 2008, 2012). This property is shared by the NaV1.1+/− horizontal slices in which we have observed RMDs. However, RMDs in our experiments have been observed only in NaV1.1+/− slices, they are generated only in the epileptic period, they can be in some cases long enough to be considered ictal-like activities, and standard 4AP ictal-like activities can be generated in the neocortex in slices that generate hippocampal RMDs. Moreover, they are similar to the recurrent epileptiform activity observed in the entorhinal cortex during combined application of 4-AP and bicuculline, and to the late recurrent discharges observed after prolonged exposure to low Mg2+ artificial cerebrospinal fluid (ACSF) (Pfeiffer et al., 1996; Bruckner et al., 1999). Of interest, these activities are generated in conditions of reduced GABAergic transmission, similarly to RMDs in NaV1.1+/− slices, and are resistant to classic anticonvulsants. Conversely, typical 4-AP ictal-like discharges have been proposed to be generated by GABAergic activities (which are reduced in NaV1.1+/− slices) and can be blocked by several classic anticonvulsants (Bruckner et al., 1999; Barbarosie et al., 2002). Therefore, we propose that RMDs can be considered a specific epileptiform activity of NaV1.1+/− slices, which highlights the important role of the hippocampus in this mouse model of DS.
Depth electrode recordings during hyperthermia-induced seizures in vivo confirmed that the hippocampus is involved in the generation of ictal activity, and showed prominent hippocampal activity during early preictal phases in comparison with the somatosensory cortex. Although the accurate determination of brain areas particularly involved in seizure generation would require recordings with more electrodes, our results are consistent with the in vitro data.
Moderate electric stimulations of slices could not induce epileptiform activity. However, they disclosed specific signs of network hyperexcitability in NaV1.1+/− hippocampal slices. In particular, in the epileptic period we observed an increased number of population spikes in posttetanic afterdischarges and in response to the second stimulus of paired-pulse stimulations. Of interest, these signs of hyperexcitability were reduced in slices from the preepileptic period (P14–P18), when NaV1.1+/− mice do not show behavioral seizures (Yu et al., 2006; Ogiwara et al., 2007; Oakley et al., 2009). This evidence, together with the lack of RMDs and of modifications of 4-AP–induced GABAergic activities, suggests that reduction of GABAergic inhibition is larger in the epileptic period than in the preepileptic one, although the mutation of NaV1.1 (the underlying cause of epilepsy in DS) is present also in the preepileptic period. However, NaV1.1 begins to be expressed at around P10 and shows an increase in expression level between the second and third postnatal weeks in rodents (Beckh et al., 1989; Ogiwara et al., 2007). This can be the main reason for a progressive reduction of GABAergic inhibition in NaV1.1+/− mice during development, which can lead to neuronal network hyperexcitability in the preepileptic period (between P10 and P18) and to ictal activity in the epileptic period. However, other modifications in network functions, physiologic-developmental ones, or induced by the initial network hyperexcitability, may contribute in setting the transition from the two periods.
Notably, the specific activities that we have observed in NaV1.1+/− slices may be used as in vitro targets in preclinical screening of drugs.