Watch Out,No Brakes! Impaired Inhibition Results in Hyperexcitable Networks

Impaired Action Potential Initiation in GABAergic Interneurons Causes Hyperexcitable Networks in an Epileptic Mouse Model Carrying a Human NaV1.1 Mutation

Hedrich UB, Liautard C, Kirschenbaum D, Pofahl M, Lavigne J, Liu Y, Theiss S, Slotta J, Escayg A, Dihné M, Beck H, Mantegazza M, Lerche H. J Neurosci 2014;34:14874–14889.

Mutations in SCN1A and other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of an SCN1A mouse model carrying the Na_V1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na⁺ channels in interneurons and persistent Na⁺ currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca²⁺ imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem dis-inhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation.

Commentary

Generalized/genetic epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome that is characterized by phenotypic variability. Individuals with GEFS+ develop febrile seizures in early childhood that often persist beyond 6 years of age. The clinical phenotype ranges from mild febrile seizures and febrile seizures plus, to afebrile seizures with atonic, myoclonic, or absence seizures (1, 2). GEFS+ is typically associated with missense mutations in SCN1A that encode Na_V1.1 voltage-gated sodium channels with subtle biophysical defects. In contrast, mutations in SCN1A that result in loss of Na _V1.1 function cause Dravet syndrome, a severe epileptic encephalopathy characterized by a variety of seizure types with accompanying delay of psychomotor and cognitive development (3).

In the current study, Hedrich and colleagues performed a thorough neurophysiological characterization of a GEFS+ mouse engineered to carry the human mutation SCN1A-R1648H, which has been shown to have defects in fast inactivation and increased persistent sodium current when expressed in heterologous systems. Previous work showed that the Scn1a^RH mouse model exhibits infrequent spontaneous seizures and reduced threshold to seizures induced by hyperthermia and chemoconvulsants (4). To more fully understand the physiological consequences of a missense sodium channel mutation, the authors probed intrinsic firing properties of inhibitory and excitatory neurons isolated at postnatal day 14 to 20 from three brain regions typically strongly associated with epilepsy: the thalamus, cortex, and hippocampus.

A major question of this study is whether expression of the R1648H mutation results in reduced excitability of GABAergic interneurons similar to that observed for Dravet syndrome mice, or increased excitability of excitatory neurons within the thalamus, cortex, or hippocampus (5, 6). Hedrich and colleagues observed that inhibitory neurons within the thalamic nucleus reticularis (nRt), as well as fast-spiking (FS) interneurons located in cortical layer IV and stratum oriens of the hippocampal CA1 region, showed impaired excitability, firing few action potentials in response to a depolarizing current injection. However, excitatory cells within the thalamus, cortical layer V, and hippocampal stratum pyramidale were all similar for WT, Scn1a^RH/+ and Scn1a^RH/RH mice. This suggests loss of function of GABAergic neurons may be a common mechanism of SCN1A-related epilepsies.

Previous voltage-clamp recording of Na_V1.1-R1648H heterologously expressed in Xenopus oocytes or tsA201 cells showed reduced current density and increased persistent current, as well as defects in recovery from inactivation and voltage-dependence of slow inactivation (7, 8). To determine if dysfunction of GABAergic neurons is caused by abnormal somatic sodium current resulting from the mutation, the authors performed voltage-clamp recording of nucleated patches in acute brain slices from inhibitory neurons in the CA1 stratum oriens of WT, Scn1a^RH/+, and Scn1a^RH/RH mice. In contrast to the heterologous cell data, no differences were observed in current density, voltage dependence of activation or fast inactivation, or recovery from inactivation between WT, Scn1a^RH/+, or Scn1a^RH/RH mice. Further, the authors did not observe increased persistent current either in nRt or hippocampal inhibitory neurons. These data suggest that defects in GABAergic activity do not result from defects in somatic sodium current. Upon analysis of action potential phase plots—which reveal both the axonal and somatic components of action potential initiation and propagation—Hedrich and colleagues found that hippocampal FS neurons from Scn1a^RH/+ and Scn1a^RH/RH animals showed a significant impairment of action potential initiation, as well as a decrease in membrane potential acceleration, which is consistent with defects within the axon initial segment, where Na_V1.1 is expressed at high density.

To examine the impact of reduced excitability of thalamic and cortical interneurons, the authors recorded spontaneous inhibitory postsynaptic currents (sIPSCs) in different populations of neurons of the thalamocortical circuit receiving inhibitory input including cortical layer V pyramidal cells, reciprocally self-inhibiting nRt neurons, and excitatory thalamocortical relay neurons. All three neuronal subtypes from Scn1a^RH/+ animals showed reduced frequencies of sIPSCs compared to WT animals, consistent with a loss of GABAergic tone. As an important control, when action potentials were inhibited with the specific sodium channel blocker tetrodotoxin, no differences were detected in the frequency of sIPSCs. These data, in addition to the finding that sIPSC amplitude was not different between WT and Scn1a^RH mice, suggest that GABAergic synapses are fully intact and that the reduced excitability of inhibitory neurons is a result of defective action potential initiation and propagation.

Finally, the authors used three different methods to examine network activity in Scn1a^RH mice. A first screen was performed using primary hippocampal neuronal cultures from either WT or Scn1a^RH/+ mice plated onto a 60-channel multi-electrode array (MEA), in which the activity of neurons at each channel could be assessed simultaneously. They observed that compared to WT neurons, Scn1a^RH/+ neurons showed periods of low activity and some longer periods of highly synchronous activity, while WT animals showed a larger number of bursts of shorter duration. To determine if these differences were due to defects in GABAergic tone, the authors applied the GABA antagonist bicuculline and found that GABA receptor inhibition had a larger impact on WT neurons, inducing higher synchrony and reducing the number of bursts to a level similar to that observed for heterozygous animals. Consistent with defects in GABAergic activity of Scn1a^RH/+ neurons, little effect was observed with further reduction of GABAergic receptor activity by inhibition with bicuculline.

As a second test of network activity, Hedrich and colleagues used extracellular field recording of layer IV and V neocortical S1 region, and in the thalamic nRt and ventrobasal (VB) regions. While no differences were observed between WT and Scn1a^RH/+ animals when network activity was stimulated in the VB and recorded in layer IV cortical region, they did observe that spontaneous neuronal discharges in Scn1a^RH/+ slices were more frequent than those in slices from WT animals. Additionally, slices from Scn1a^RH/+ animals exhibited high frequency oscillations within the pathophysiological range of 200 to 600Hz, which were absent in WT controls. Third, using multineuron Ca²⁺ imaging, they observed increased spontaneous activity in both Scn1a^RH/+ and Scn1a^RH/RH mice compared to WT animals, further suggestive of defective neuronal networks.

Overall, this study is a step toward understanding the underlying mechanism of GEFS+ and suggests that there may be a common pathophysiology for SCN1A-related epilepsies. Like Scn1a^+/− Dravet syndrome mice, Scn1a^RH mice showed reduced excitability of GABAergic interneurons from several brain regions. While the biophysical defects associated with the R1648H mutation as determined by heterologous expression may not have been observed in neurons from these animals, the defects in excitability, action potential initiation, propagation, and network activity are clear. Further experiments are needed to determine how mutations in SCN1A that result in impaired GABAergic neuronal activity can result in epilepsies as divergent as Dravet syndrome and GEFS+.