Nav1.1gating Against the Current: Ndnf Interneurons Hold Strong in Dravet Syndrome

Abstract

Ndnf Interneuron Excitability Is Spared in a Mouse Model of Dravet Syndrome

Liebergall SR, Goldberg EM. J Neurosci. 2024;44(17):e1977232024. doi: 10.1523/JNEUROSCI.1977-23.2024. PMID: 38443186; PMCID: PMC11044195

Dravet syndrome (DS) is a neurodevelopmental disorder characterized by epilepsy, developmental delay/intellectual disability, and features of autism spectrum disorder, caused by heterozygous loss-of-function variants in SCN1A encoding the voltage-gated sodium channel α subunit Nav1.1. The dominant model of DS pathogenesis is the “interneuron hypothesis,” whereby GABAergic interneurons (INs) express and preferentially rely on Nav1.1-containing sodium channels for action potential (AP) generation. This has been shown for three of the major subclasses of cerebral cortex GABAergic INs: those expressing parvalbumin (PV), somatostatin, and vasoactive intestinal peptide. Here, we define the function of a fourth major subclass of INs expressing neuron-derived neurotrophic factor (Ndnf) in male and female DS (Scn1a+/−) mice. Patch-clamp electrophysiological recordings of Ndnf-INs in brain slices from Scn1a+/– mice and wild-type (WT) controls reveal normal intrinsic membrane properties, properties of AP generation and repetitive firing, and synaptic transmission across development. Immunohistochemistry shows that Nav1.1 is strongly expressed at the axon initial segment (AIS) of PV-expressing INs but is absent at the Ndnf-IN AIS. In vivo two-photon calcium imaging demonstrates that Ndnf-INs in Scn1a+/− mice are recruited similarly to WT controls during arousal. These results suggest that Ndnf-INs are the only major IN subclass that does not prominently rely on Nav1.1 for AP generation and thus retain their excitability in DS. The discovery of a major IN subclass with preserved function in the Scn1a+/− mouse model adds further complexity to the “interneuron hypothesis.”

Commentary

Dravet syndrome (DS) is a severe form of lifelong epilepsy that affects 1:15,700 infants born in the U.S. every year. ¹ Epileptic seizures in DS are early onset, difficult to control, and accompanied by comorbidities such as developmental delay, cognitive and motor impairment, and a high risk of sudden unexpected death in epilepsy. In 90% of cases, the root cause of DS is a loss-of-function mutation in SCN1A, the gene encoding the α subunit of Nav1.1, a voltage-gated sodium channel. Nav1.1 is expressed predominantly in the central nervous system, and there, mostly in GABAergic inhibitory interneurons (INs). Indeed, parvalbumin (PV)-, somatostatin (SST)-, and vasoactive intestinal peptide (VIP)-expressing GABAergic INs, the three major subclasses of INs in the brain, all heavily rely on the expression of Nav1.1 in the axon initial segment (AIS) for action potential (AP) generation.^2–4 Therefore, in DS, a reduction in Nav1.1 is thought to cause decreased firing in GABAergic INs as one of the key biological processes leading to DS pathophysiology. ⁵ In a recent publication in the Journal of Neuroscience, ⁶ Sophie Liebergall and Ethan Goldberg refer to this concept as the “interneuron hypothesis” and argue that it oversimplifies the underlying mechanisms by showing that a fourth major subclass of GABAergic INs that expresses neuron-derived neurotrophic factor (Ndnf) is mostly resilient to reduced firing in a Scn1a^+/– mouse model of DS.

While PV, SST, and VIP INs have been studied in DS models, this study is the first to provide a detailed and elegant electrophysiological analysis of Ndnf INs. Using patch-clamp electrophysiology in acute brain slices, they found that the passive and active electrical properties of Ndnf INs were mostly unaffected in both male and female Scn1a^+/– mice at postnatal days P16–21, an age at which other interneuron types displayed reduced AP firing. This finding suggests that Ndnf INs do not rely heavily on Nav1.1 to fire action potentials, consistent with the observation that, unlike other GABAergic INs, Scn1a^+/+ Ndnf INs lacked detectable Nav1.1 immunohistochemical signal in the AIS. Furthermore, paired recordings between Ndnf INs and Layer 2/3 pyramidal neurons, onto which Ndnf neurons synapse, found no difference in synaptic function in P16–21 slices between Scn1a^+/– and age-matched controls, suggesting that the output function of Scn1a^+/– Ndnf INs is normal. Ndnf interneuron function in vivo also appeared to be unaffected in Scn1a^+/− mice. Using two-photon calcium imaging in the somatosensory cortex of awake behaving mice, the authors found that the activity of Ndnf INs was normal during transitions from quiet wakefulness to arousal at a time when locomotion speed and pupil diameter increased—a transition that is known to normally involve Ndnf INs. ⁷ Together, these findings suggest that Ndnf INs are less sensitive to loss of Scn1a than are other INs.

Are Ndnf INs entirely resilient in DS? Although their electrical properties were mainly unaffected, their AP rise time was slightly prolonged and the maximal steady-state frequency was decreased in Scn1^+/– mice, which would be consistent with some role of Nav1.1 in these cells. Of note, expression levels of Scn1a transcripts are comparable between Ndnf and other interneuron subclasses. ⁸ Given that Nav1.1 was not detected in the AIS of Ndnf INs and that action potentials were only modestly affected, then the precise role of Nav1.1 in these cells remains to be determined. Indeed, anecdotal evidence cited in a review^5,9 suggested that deleting Scn1a in serotonin receptor 5-HT3aR-expressing cells, which include Ndnf INs, produced mild autistic behavior but not epilepsy. Future studies specifically deleting Scn1a in Ndnf INs would be necessary to determine the contribution of Ndnf cells to DS phenotypes.

In sum, the authors show that Ndnf INs are less sensitive to Scn1a haploinsufficiency than other INs, which adds to growing evidence for a more complex view of INs’ vulnerability in DS. Indeed, other studies have shown that certain aspects of the DS pathophysiology may result from changes other than just the simple loss of Scn1a in GABAergic INs. First, although PV INs show reduced firing at earlier stages of development (P11–21) in the Scn1a^+/– mice, Goldberg and colleagues ¹⁰ have shown that at P35 their firing properties are restored even though mice continue to seize at this age, suggesting that reduced firing in PV INs is not required for the chronic epilepsy phenotype of adult DS mice. Second, GABAergic neurons in the reticular thalamus that also rely on Nav1.1 for AP firing become hyperexcitable in the Scn1a^R1407+/– model of DS, rather than reducing their firing activity. ¹¹ Their enhanced firing, due to compensatory changes, is responsible for nonconvulsive thalamocortical seizures which are one of the many facets of DS. ¹¹ Third, excitatory neurons express Scn1a too. For instance, in the hippocampus, excitatory neurons upregulate sodium currents in Scn1a^+/– mice at P21–24 and start to fire spontaneously. This firing correlates with the age-dependent onset of lethality in mice. ¹² Similarly, excitatory neurons differentiated from induced pluripotent stem cells derived from DS patients with SCN1A haploinsufficiency also showed increased sodium currents. ¹³

This new study by Liebergall and Goldberg adds nuance to the hypothesis that DS pathophysiology is simply caused by decreased GABAergic interneuron firing by providing compelling evidence that Ndnf INs are more resilient in DS mice than the other major subtypes of INs, and thus unlikely to be key drivers of DS. More generally, this study reminds us that to fully understand the multiple facets of DS pathophysiology, it will be important to consider cell-type heterogeneity and cell-specific vulnerability and to carefully assess the role of compensatory mechanisms, either adaptive or maladaptive, to Scn1a deficiency.