“It Was the Interneuron With the Parvalbumin in the Hippocampus!” “No, It Was the Pyramidal Cell With the Glutamate in the Cortex!” Searching for Clues to the Mechanism of Dravet Syndrome – The Plot Thickens

Commentary

Strain- and Age-Dependent Hippocampal Neuron Sodium Currents Correlate With Epilepsy Severity in Dravet Syndrome Mice.

Mistry AM, Thompson CH, Miller AR, Vanoye CG, George AL Jr, Kearney JA. Neurobiol Dis 201;65:1–1124434335.

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a+/−) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a+/− mice on strain 129S6/SvEvTac (129.Scn1a+/−) have no overt phenotype and normal survival compared with Scn1a+/− mice bred to C57BL/6J (F1.Scn1a+/−) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21–24 (P21–24) 129.Scn1a+/− or F1.Scn1a+/− mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a+/− mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a+/− mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a+/− and F1.Scn1a+/− mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a+/− neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a+/− animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a+/− mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21–24 correlates with age-dependent onset of lethality in F1.Scn1a+/− mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

Dravet syndrome (DS, also known as Severe Myoclonic Epilepsy of Infancy) is a catastrophic pediatric epileptic encephalopathy with cognitive, behavioral, and motor impairments, as well as a high risk of sudden unexpected death in epilepsy (SUDEP). The first mutations linked to DS were identified in SCN1A, the gene encoding the a subunit of the voltage-gated sodium channel Na_V1.1 (1). To date, the majority of DS patients have de novo SCN1A mutations that result in haploinsufficiency (2). This raises some interesting questions: How does reduced expression of a major brain sodium channel gene result in hyperexcitability and network synchrony? Further, why do individuals with the same heterozygous SCN1A mutation, even within the same family, sometimes have divergent seizure phenotypes?

A major breakthrough in understanding the mechanism of DS occurred with the development of the first mouse model of the disease by the Catterall group (3). Global deletion of Scn1a yielded a strong seizure phenotype in heterozygous animals and was the first in vivo confirmation of Scn1a haploinsufficiency resulting in seizures. Patch clamp recordings in acutely dissociated hippocampal neurons isolated from postnatal day (P) 14–16 Scn1a^+/− mice demonstrated that morphologically identified bipolar neurons, but not pyramidal neurons, had dramatically reduced sodium current density. This resulted in reduced firing frequency of interneurons in response to injected current. Nav1.1 was postulated to be the major functional sodium channel in interneurons, particularly in fast-spiking, parvalbumin-positive cells (3, 4). In contrast, glutamatergic pyramidal neurons were proposed to rely on other brain sodium channels, possibly Nav1.2 or Nav1.6, for firing. Based on this model, it was hypothesized that pathophysiology in DS is due to dysfunctional inhibitory circuits: the “interneuron hypothesis.”

Since the initial description of Scn1a^+/− mice by the Catterall group, two competing theories have emerged to explain the mechanism of DS. The interneuron hypothesis is largely supported by work done in mouse models (4–10), while more recent data that challenge it, showing increased sodium current density and hyperexcitability in both inhibitory and excitatory neurons, have been generated using human DS patient-derived induced pluripotent stem cell (iPSC) neurons (11, 12). Is it possible that this discordance results from differences in the model systems themselves? In their paper in Neurobiology of Disease, Mistry and colleagues begin to bridge the gap between competing DS mechanistic theories. Prior work from Dr. Kearney's laboratory (13) showed that phenotypic severity of the Scn1a^+/− mutation in mice is highly dependent on genetic background. Scn1a^+/− mice maintained on the 129 strain exhibited no seizures and lived normal life spans. In contrast, crossing Scn1a^+/− mice on the 129 background to the C57Bl/6J strain, referred to as F1.Scn1a^+/−, resulted in animals with a severe phenotype, including overt seizures and SUDEP. Thus, genetic background is key to the severity of pathophysiology in DS mouse models. In the Mistry et al. study, the authors compare sodium current properties and neuronal firing in acutely dissociated hippocampal neurons from Scn1a^+/− mice on the 129 versus the 129 x C57Bl/6J F1 backgrounds at two different developmental time points. In all of their experiments, the identity of acutely dissociated bipolar interneurons was confirmed by single cell RT-PCR. Using the criteria of Gad67 but not Gfap positivity, only 48% of morphologically identified “bipolar” neurons were confirmed as such. This is an important consideration for the interpretation of previous work in which a proportion of morphologically identified “bipolar neurons” may have been, in reality, astrocytes.

Patch clamp recording of hippocampal neurons showed that sodium current density was significantly lower in identified P21-24 GABAergic interneurons from the phenotypically more severe F1.Scn1a^+/− strain compared to wild-type littermates, similar to the Catterall et al. (3) study focusing on neurons isolated from P14-16 Scn1a^+/− mice bred for 6 generations onto C57Bl/6 from 129SvJ. In contrast, GABAergic interneuron sodium current densities in 129.Scn1a^+/− mice (which had no spontaneous seizures) and their corresponding wild-type littermates were indistinguishable. Surprisingly, and similar to more recently published findings in human iPSC forebrain neuron models of DS (11), sodium current density was elevated in pyramidal neurons from both P21–24 129.Scn1a^+/− and F1.Scn1a^+/− mice compared to strain-matched wild-type littermates. Importantly, pyramidal neurons from the phenotypically more severe F1.Scn1a^+/− strain displayed a significantly larger increase in sodium current density compared to pyramidal neurons from the less severe 129.Scn1a^+/− strain. In addition, F1.Scn1a^+/−, but not 129.Scn1a^+/− P21–24 pyramidal neurons exhibited a significant hyperpolarizing shift in the voltage-dependence of activation compared to wild-type. Consistent with this observation and in agreement with previous studies (8), P21–24 F1.Scn1a^+/− pyramidal neurons exhibited high frequency spontaneous firing and greater excitability across a range of current injections. P21–24 pyramidal neurons from the less phenotypically severe 129.Scn1a^+/− strain also exhibited higher spontaneous firing frequencies than wild-type, but their rate of firing was significantly less than that observed in the F1.Scn1a^+/− animals.

These observed increases in pyramidal neuron sodium current density were age-dependent, occurring later in development than the observed reduction in inhibitory neuron sodium current density. Sodium current densities in P14–15 pyramidal neurons were not significantly different between mutant and wild-type on either background. Interestingly, the exaggerated increase in sodium current density in the P21–24 F1.Scn1a^+/− excitatory pyramidal neurons correlated with age-dependent onset of lethality. Finally, the authors reported strain-dependent trends in compensatory up- or down-regulation of other brain sodium channel transcripts in response to Scn1a haploinsufficiency, results that may shed light on patient-specific difference in phenotype severity.

This study provides important new clues to solving the mechanism of DS. First, it is now clear from multiple models of DS that both inhibitory and excitatory neurons have altered sodium currents, suggesting that both inhibitory and excitatory networks contribute to seizure severity. Inhibitory neuron sodium channel loss-of-function combined with excitatory neuron sodium channel gain-of-function may result in an inhibition-excitation imbalance during the P21–24 developmental period in F1.Scn1a^+/− mice leading to severe seizures. Second, similar to human DS patients with variable seizure severity and susceptibility to SUDEP, the phenotypic severity of DS mouse models is highly dependent on genetic background.

In spite of these advances, important questions remain. A detailed developmental time course of sodium channel protein expression in excitatory versus inhibitory neurons (and in different interneuronal subtypes) in specific brain areas and comparing mouse strains is a necessary, yet difficult, next step. A detailed examination of sodium channel expression in the developmental period surrounding P18, the time point at which F1.Scn1a^+/− begin to seize, would be particularly informative. Not only sodium channel pore-forming a subunits, but also ß subunits, need to be investigated. It will be important to know whether impaired inhibitory neuron GABAergic signaling through loss of Nav1.1-mediated sodium current density during early development (e.g., P14–16) can subsequently influence sodium current expression in excitatory neurons, presumably through synaptic signaling, at later time points (P21–24). If the expression of other sodium channel subunits is indeed altered in response to Scn1a haploinsufficiency, as suggested here and in the human iPSC neuron models, what is underlying the mechanism for this compensatory gene expression? Does the developmental switch of early GABAergic excitatory to inhibitory signaling occur normally in Scn1a^+/− mouse and iPSC neuron models? If not, this in itself changes the prevailing ideas surrounding disinhibition. Finally, what other genes—that is, encoding other ion channels, cell adhesion molecules, or synaptic proteins—are altered in response to Scn1a haploinsufficency? New animal models, as well as analysis of DS patient-derived iPSC neurons, which allow correlation of human epilepsy phenotypes with cell autonomous electrophysiological, genetic, and channel protein complex characterization, will shed further light on this rapidly progressing field.