TrkB hyperactivity contributes to brain dysconnectivity, epileptogenesis, and anxiety in zebrafish model of Tuberous Sclerosis Complex

Abstract

Tuberous Sclerosis Complex (TSC) is a rare genetic disease that manifests with early symptoms, including cortical malformations, childhood epilepsy, and TSC-associated neuropsychiatric disorders (TANDs). Cortical malformations arise during embryonic development and have been linked to childhood epilepsy before, but the underlying mechanisms of this relationship remain insufficiently understood. Zebrafish have emerged as a convenient model to study elementary neurodevelopment; however, without in-depth functional analysis, the Tsc2-deficient zebrafish line cannot be used for studies of TANDs or new drug screening. In this study, we found that the lack of Tsc2 in zebrafish resulted in heterotopias and hyperactivation of the mTorC1 pathway in pallial regions, which are homologous to the mammalian cortex. We observed commissural thinning that was responsible for brain dysconnectivity, recapitulating TSC pathology in human patients. The lack of Tsc2 also delayed axonal development and caused aberrant tract fasciculation, corresponding to the abnormal expression of genes involved in axon navigation. The mutants underwent epileptogenesis that resulted in nonmotor seizures and exhibited increased anxiety-like behavior. We further mapped discrete parameters of locomotor activity to epilepsy-like and anxiety-like behaviors, which were rescued by reducing tyrosine receptor kinase B (TrkB) signaling. Moreover, in contrast to treatment with vigabatrin and rapamycin, TrkB inhibition rescued brain dysconnectivity and anxiety-like behavior. These data reveal that commissural thinning results in the aberrant regulation of anxiety, providing a mechanistic link between brain anatomy and human TANDs. Our findings also implicate TrkB signaling in the complex pathology of TSC and reveal a therapeutic target.

stim2b Knockout Induces Hyperactivity and Susceptibility to Seizures in Zebrafish Larvae

“…This experiment was performed as described previously [33]. Two minutes before recording locomotor activity, the larvae were transferred to a Petri dish.…”

“…To further investigate anxiety-like behavior in stim2b −/− zebrafish, the light preference test was performed as described previously [32,33] (Figure 4A). Zebrafish larvae exhibited a tendency to stay in the light part and actively avoid the dark part [30].…”

“…Studying zebrafish behavior provides vast possibilities even during the larval stage (reviewed in [28,29]). Assays that enable assessments of anxiety levels, such as thigmotaxis [30,31] and phototaxis [32,33], have been well established in this model. Zebrafish are also often used for drug screening, and several neuroactive compounds have been tested in this animal [28,34].…”

Knockout of stim2a Increases Calcium Oscillations in Neurons and Induces Hyperactive-Like Phenotype in Zebrafish Larvae

“…Thus, complex behavior cannot be analyzed in S tim2 knockout mice [ 31 , 32 ]. In zebrafish, the nervous system develops at an early stage and allows larvae to exhibit complex behavior [ 42 , 50 , 59 ]. Additionally, because of larval transparency, in vivo Ca 2+ measurements can be performed in neurons [ 45 , 60 ].…”

“…The stim2a −/− larvae reacted to changes in light in the visual–motor response test, thus excluding the possibility of visual deficits in the mutant animals. Lower light preference has been linked to a decrease in the anxiety in zebrafish larvae [ 50 , 59 , 61 ]. Such unpredictability between phototaxis and thigmotaxis indicates the involvement of different brain regions that control these two processes [ 51 , 62 ].…”