Engineering Synthetic circRNAs for Efficient CNS Expression.

Circular RNAs (circRNAs) have recently emerged as a promising modality for gene and RNA-based therapies. They are more stable than their linear counterpart and can be designed for efficient expression in different cell and tissue types. In this chapter, we developed different backsplicing circRNA cassettes that can enable efficient gene expression in various cell and tissue types. Furthermore, we packaged cassettes encoding circRNAs into adeno-associated viral (AAV) vectors that can be delivered via intracerebroventricular (ICV) injections to achieve expression in murine brain tissue. We provide detailed methods for the design of backsplicing circRNAs, circRNA detection, and generation of AAV-circRNA vectors for CNS dosing and expression in mice.

Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing.

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5' and 3' segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

Socially Mediated Shift in Neural Circuits Activation Regulated by Synergistic Neuromodulatory Signaling.

Animals exhibit context-dependent behavioral decisions that are mediated by specific motor circuits. In social species these decisions are often influenced by social status. Although social status-dependent neural plasticity of motor circuits has been investigated in vertebrates, little is known of how cellular plasticity translates into differences in motor activity. Here, we used zebrafish (Danio rerio) as a model organism to examine how social dominance influences the activation of swimming and the Mauthner-mediated startle escape behaviors. We show that the status-dependent shift in behavior patterns whereby dominants increase swimming and reduce sensitivity of startle escape while subordinates reduce their swimming and increase startle sensitivity is regulated by the synergistic interactions of dopaminergic, glycinergic, and GABAergic inputs to shift the balance of activation of the underlying motor circuits. This shift is driven by socially induced differences in expression of dopaminergic receptor type 1b (Drd1b) on glycinergic neurons and dopamine (DA) reuptake transporter (DAT). Second, we show that GABAergic input onto glycinergic neurons is strengthened in subordinates compared with dominants. Complementary neurocomputational modeling of the empirical results show that drd1b functions as molecular regulator to facilitate the shift between excitatory and inhibitory pathways. The results illustrate how reconfiguration in network dynamics serves as an adaptive strategy to cope with changes in social environment and are likely conserved and applicable to other social species.

The mechanisms shaping CA2 pyramidal neuron action potential bursting induced by muscarinic acetylcholine receptor activation.

Recent studies have revealed that hippocampal area CA2 plays an important role in hippocampal network function. Disruption of this region has been implicated in neuropsychiatric disorders. It is well appreciated that cholinergic input to the hippocampus plays an important role in learning and memory. While the effect of elevated cholinergic tone has been well studied in areas CA1 and CA3, it remains unclear how changes in cholinergic tone impact synaptic transmission and the intrinsic properties of neurons in area CA2. In this study, we applied the cholinergic agonist carbachol and performed on-cell, whole-cell, and extracellular recordings in area CA2. We observed that under conditions of high cholinergic tone, CA2 pyramidal neurons depolarized and rhythmically fired bursts of action potentials. This depolarization depended on the activation of M1 and M3 cholinergic receptors. Furthermore, we examined how the intrinsic properties and action-potential firing were altered in CA2 pyramidal neurons treated with 10 µM carbachol. While this intrinsic burst firing persisted in the absence of synaptic transmission, bursts were shaped by synaptic inputs in the intact network. We found that both excitatory and inhibitory synaptic transmission were reduced upon carbachol treatment. Finally, we examined the contribution of different channels to the cholinergic-induced changes in neuronal properties. We found that a conductance from Kv7 channels partially contributed to carbachol-induced changes in resting membrane potential and membrane resistance. We also found that D-type potassium currents contributed to controlling several properties of the bursts, including firing rate and burst kinetics. Furthermore, we determined that T-type calcium channels and small conductance calcium-activated potassium channels play a role in regulating bursting activity.

Effects of Social Experience on the Habituation Rate of Zebrafish Startle Escape Response: Empirical and Computational Analyses.

While the effects of social experience on nervous system function have been extensively investigated in both vertebrate and invertebrate systems, our understanding of how social status differentially affects learning remains limited. In the context of habituation, a well-characterized form of non-associative learning, we investigated how the learning processes differ between socially dominant and subordinate in zebrafish (Danio rerio). We found that social status and frequency of stimulus inputs influence the habituation rate of short latency C-start escape response that is initiated by the Mauthner neuron (M-cell). Socially dominant animals exhibited higher habituation rates compared to socially subordinate animals at a moderate stimulus frequency, but low stimulus frequency eliminated this difference of habituation rates between the two social phenotypes. Moreover, habituation rates of both dominants and subordinates were higher at a moderate stimulus frequency compared to those at a low stimulus frequency. We investigated a potential mechanism underlying these status-dependent differences by constructing a simplified neurocomputational model of the M-cell escape circuit. The computational study showed that the change in total net excitability of the model M-cell was able to replicate the experimental results. At moderate stimulus frequency, the model M-cell with lower total net excitability, that mimicked a dominant-like phenotype, exhibited higher habituation rates. On the other hand, the model with higher total net excitability, that mimicked the subordinate-like phenotype, exhibited lower habituation rates. The relationship between habituation rates and characteristics (frequency and amplitude) of the repeated stimulus were also investigated. We found that habituation rates are decreasing functions of amplitude and increasing functions of frequency while these rates depend on social status (higher for dominants and lower for subordinates). Our results show that social status affects habituative learning in zebrafish, which could be mediated by a summative neuromodulatory input to the M-cell escape circuit, which enables animals to readily learn to adapt to changes in their social environment.

Social Status-Related Differences in Motor Activity Between Wild-Type and Mutant Zebrafish.

Use of zebrafish as a model organism in biomedical research has led to the generation of many genetically modified mutant lines to investigate various aspects of developmental and cellular processes. However, the broader effects of the underlying mutations on social and motor behavior remain poorly examined. Here, we compared the dynamics of social interactions in the Tüpfel long-fin nacre mutant line, which lacks skin pigmentation, to wild-type zebrafish; and we determined whether status-dependent differences in escape and swimming behavior existed within each strain. We show that despite similarities in aggressive activity, Tüpfel long-fin nacre pairs exhibit unstable social relationships characterized by frequent reversals in social dominance compared to wild-type pairs. The lack of strong dominance relationships in Tüpfel long-fin nacre pairs correlates with weak territoriality and overlapping spatial distribution of dominants and subordinates. Conversely, wild-type dominants displayed strong territoriality that severely limited the movement of subordinates. Additionally, the sensitivity of the startle escape response was significantly higher in wild-type subordinates compared to dominants. However, status-related differences in sensitivity of escape response in Tüpfel long-fin nacre pairs were absent. Finally, we present evidence suggesting that these differences could be a consequence of a disruption of proper visual social signals. We show that in wild-type pairs dominants are more conspicuous, and that in wild-type and Tüpfel long-fin nacre pairings wild-type fish are more likely to dominate Tüpfel long-fin nacres. Our results serve as a cautionary note in research design when morphologically engineered zebrafish for color differences are utilized in the study of social behavior and central nervous system function.