Large‐scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage‐gated sodium channel isoform 1.2 (Nav1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a+/−) results in mild behavior abnormalities. The Nav1.2 expression level in Scn2a+/− mice is reported to be around 50–60% of the wild‐type (WT) level, which indicates that a close to 50% reduction of Nav1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene‐trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2agtKO/gtKO) that can survive to adulthood, with about a quarter of Nav1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2agtKO/gtKO mice. Notably, Scn2agtKO/gtKO mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety‐like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2agtKO/gtKO mice have increased fix‐pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene‐trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.

The δ-opioid receptor (δOR) has been considered as a therapeutic target in multiple neurological and neuropsychiatric disorders particularly as δOR agonists are deemed safer alternatives relative to the more abuse-liable µ-opioid receptor drugs. Clinical development of δOR agonists, however, has been challenging in part due to the seizure-inducing effects of certain δOR agonists. Especially agonists that resemble the δOR-selective agonist SNC80 have well-established convulsive activity. Close inspection suggests that many of those seizurogenic δOR agonists efficaciously recruit β-arrestin, yet surprisingly, SNC80 displays enhanced seizure activity in β-arrestin 1 knockout mice. This finding led us to hypothesize that perhaps β-arrestin 1 is protective against, whereas β-arrestin 2 is detrimental for δOR-agonist-induced seizures. To investigate our hypothesis, we characterized three different δOR agonists (SNC80, ADL5859, ARM390) in cellular assays and in vivo in wild-type and β-arrestin 1 and β-arrestin 2 knockout mice for seizure activity. We also investigated downstream kinases associated with β-arrestin-dependent signal transduction. We discovered that δOR agonist-induced seizure activity strongly and positively correlates with β-arrestin 2 efficacy for the agonist, but that indirect inhibition of ERK activation using the MEK inhibitor SL327 did not inhibit seizure potency and duration. Inhibition of the PI3K/AKT/mTOR signaling with honokiol but not PQR530, attenuated SNC80 seizure duration in β-arrestin 1 knockout, but honokiol did not reduce SNC80-induced seizures in wild-type mice. Ultimately, our results indicate that β-arrestin 2 is correlated with δOR agonist-induced seizure intensity, but that global β-arrestin 1 knockout mice are a poor model system to investigate their mechanism of action.

Arrestins are a family of proteins involved in regulation of G‐protein coupled receptor (GPCR) signaling. By binding to phosphorylated receptors, they uncouple the receptor from its corresponding G‐protein, terminating GPCR signaling in a process known as desensitization. Within the arrestin family, the β‐arrestin 1and β‐arrestin 2 isoforms are ubiquitously expressed throughout the body, including the central nervous system, and interact with a multitude of GPCRs. Additionally, the β‐arrestin proteins also have an important impact on behavior, with various studies indicating each isoform has unique signaling interactions and different roles in drug responses. This has prompted researchers to develop molecules that are specific towards recruitment of one isoform over the other; as well as further understand the biological role of each protein. The δ‐opioid receptor (DOR) is a promising target for the treatment of a variety of neurological disorders, including chronic pain disorders, alcohol use disorder and mood disorders. However, a major side effect that occurs when activating the DOR is the induction of seizures. Notably, mice with a genetic knockout (KO) of β‐arrestin 1 mice show a markedly increased sensitivity to agonist induced seizures compared to wild type or β‐arrestin 2 KO mice. Understanding the mechanism linking DOR agonists and seizures will allow for the use of more rational drug design to develop effective therapeutics. This project aims to 1) investigate the cellular mechanism of action for β‐arrestin's involvement in the seizurogenic effects of DOR activation 2) Assess whether DOR agonists with lower β‐arrestin recruitment efficacy are less seizurogenic and 3) Determine if β‐arrestin 1 KO mice are a novel model of status epilepticus? To address these questions, we measured seizure activity (assessed by Racine score), in male and female wild‐type, β‐arrestin 1 KO and β‐arrestin 2 KO C57BL/6 mice (n≥6 per treatment). We utilized three different DOR agonists, which we determined differed in their β‐arrestin recruitment efficacy using cellular signaling assays. When we injected these three agonists, SNC80, ADL5859 and ARM390 at equi‐analgesic doses, we noted a strong correlation between β‐arrestin 2 recruitment efficacy and seizure intensity. We did not observe any sex differences. In the hippocampus of our mice we also noted a strong increase in pERK following DOR activation. The ERK activation was more pronounced when mice were treated with a DOR agonist with strong β‐arrestin recruitment efficacy, and also was more pronounced in β‐arrestin 1 KO mice. Blocking ERK activation, with the MEK inhibitor SL327, however, did not reduce DOR induced seizure, suggesting that the ERK activation comes as a result of the seizure and is not part of the mechanism of action of DOR‐induced seizures. Thus far, our results suggest that the likelihood of seizures induced by DOR activation can be mitigated using a DOR agonist with low β‐arrestin 2 recruitment efficacy.