In this report, we provide the first example where knockdown of a gene known to be responsible for epilepsy in humans results in seizure-like behaviors in zebrafish. Several independent observations suggest that specific loss of lgi1a is directly responsible for this phenotype: (i) targeting lgi1a function using two different approaches (translation and splicing) results in the same phenotype; (ii) a mismatch control MO does not alter mRNA splicing or mRNA levels and does not result in seizure-like behavior; (iii) co-injection of processed lgi1a mRNA with the MO-E3 MO significantly reduces seizure-like activity. The lgi1a morphants, therefore, provide a tractable model to investigate the involvement of this and other genes in the development of seizures.
LGI1 mutations in humans predispose to epilepsy, and there is some evidence that this is due in part to impaired synaptic transmission (12
). Other evidence suggests that LGI1 may have a role in brain development (16
) but it is not known whether abnormal development underlies LGI1-linked seizure events. Imaging studies have suggested focal abnormalities in brains of some ADPEAF patients, although this phenotype appears heterogeneous. Previous in vitro
molecular studies suggested that LGI1 might affect axon guidance pathways (21
), which would have a consequence for normal brain development. Abnormal axonal targeting, if it resulted in failed synapse formation, for example, could lead to apoptosis in neurons. Increased apoptosis was observed in the brains of fish with loss of lgi1a
function. In addition, hypocellularity in the brain of lgi1a
morphants supports the idea that LGI1 may play a fundamental role in embryonic brain development in vertebrates.
Although hyperactivity was a common phenotype in Lgi1a morphants and PTZ-treated zebrafish, there were clearly behavioral differences between the two groups. These differences could depend on protocol differences such as the age of the fish at the time of treatment as well as the relative doses of PTZ used. In our study, we first detected increased hyperactivity using 2.5 mm
PTZ, which was also reported by Baraban et al
), although no further details were given at this dose. Instead, this and other studies (24
) routinely used doses that were either 15 or 20 mm
, in order to accentuate the seizure response in a short time frame. This high dose, therefore, could produce a phenotype severity beyond that induced by lgi1a
knockdown. By comparison, the Lgi1a morphants only demonstrated a behavior that would have been scored between stages 1 and 2 in PTZ-treated fish, resembling the rapid swimming/whirlpool phenotype. Of particular relevance to Lgi1a morphants, however, was the phenotype reported in the Xenopus
) which, even though 15 mm
PTZ was used, was described as ‘swimming in tight circles’ during the earliest stage-1 phenotype. This was exactly the phenotype seen in the Lgi1a morphants. Furthermore, a C-shaped tail movement was also described in early stages of tadpole treatment, which was also seen in the Lgi1 morphants. Thus, it appears that the seizure-like phenotype in the lgi1a
morphants more closely resembles the earliest stages of PTZ-induced seizure activity, which may only be transient in the presence of high-dose PTZ. At higher MO doses, seizure-like phenotypes were more pronounced and their interpretation was complicated by the confounding observation that morphants also show pronounced tail deformities. The swimming behavior, therefore, was potentially a byproduct of improper tail development, potentially preventing conventional tail propulsion. The synergy studies, however, demonstrated that low-dose morphants with normal tail shape were more susceptible to PTZ-induced seizures, demonstrating a predisposing effect of the knockdown of lgi1a
. The hyperactivity we saw following PTZ treatment of low-dose lgi1a morphants may reflect a kindling effect that sensitized the fish to drug-induced seizures. A similar effect was seen in the recently described Lgi1 mutant null mice (14
), which showed an early onset of seizures followed by premature death, as in the zebrafish model described here. In one study (15
), heterozygous Lgi1 null mice, which did not show overt seizures, showed increased susceptibility to PTZ-induced seizures compared with wild-type littermates. In this respect, the Lgi1a morphant fish mimic the phenotype seen in Lgi1 mutant mice.
Another possible explanation for the slightly different seizure-like behavior in Lgi1a morphants and PTZ-treated zebrafish is their age at the time of exposure to the seizure-inducing agent. In all reports of PTZ-only treatment, the drug was given to 6–7-day-old fish, when the majority of morphological development and primary neurogenesis was complete. In contrast, Lgi1a morphants were treated at 3dpf in order to ensure that sensitization tests were performed at a time point where knockdown of Lgi1a expression was likely to be in effect. Therefore, it may not be surprising that the specific seizure-like behavioral phenotype was different in the two paradigms. In one report (28
), however, electrographic analysis of 3dpf fish treated with 15 mm
PTZ demonstrated seizure activity, which was less complex than that seen in 7dpf fish. The age differences, however, may be particularly relevant if low-dose lgi1a knockdown results in abnormal brain development. Notwithstanding, the similarities in seizure-like behavior, together with the kindling of PTZ-induced seizures as a result of 50% reduction of wild-type lgi1a expression, provide convincing evidence for a causative role for Lgi1a in zebrafish seizure-like behavior.
The lgi1a morphants also demonstrate that lgi1a
is important in normal embryological development in fish, since the high-dose MO-ATG morphants show severe defects in organs such as the eyes and brain and abnormal trunk development. Similar, but less severe, phenotypes were seen in the MO-E3 morphants. As expected, the penetrance of these effects is directly related to the levels of lgi1a
knockdown. As suggested in other studies (31
), we assume that the severe phenotype in the 3–4 ng MO-ATG fish is due to the fact that this MO targets both zygotic and maternal lgi1a
mRNA, creating more of an expression null in these fish, compared with the MO-E3, which only targets zygotic mRNA. This suggestion is reinforced by rescue experiments, where a preprocessed mRNA can largely overcome the consequences of knocking down de novo
Lgi1a. The spatial and temporal expression of LGI1 (29
), together with the constitutional predisposition to seizures in humans, and suggestion of reduced brain size in fish and humans having reduced lgi1a/
LGI1 expression levels, further points to the possibility that abnormal development of the brain may contribute to the seizure phenotype.
In summary, we describe a zebrafish model of seizure-like behavior as a result of the knockdown of the lgi1a gene, which is responsible for epilepsy in humans. In addition, we provide evidence that lgi1a may also play a role in normal development of zebrafish, particularly in the brain. This zebrafish model offers the opportunity to extend these observations to other genes known to interact with lgi1a/Lgi1 or involved in pathways downstream of the action of lgi1a/Lgi1.