Social Status-Dependent Shift in Neural Circuit Activation Affects Decision Making.

In a social group, animals make behavioral decisions that fit their social ranks. These behavioral choices are dependent on the various social cues experienced during social interactions. In vertebrates, little is known of how social status affects the underlying neural mechanisms regulating decision-making circuits that drive competing behaviors. Here, we demonstrate that social status in zebrafish (Danio rerio) influences behavioral decisions by shifting the balance in neural circuit activation between two competing networks (escape and swim). We show that socially dominant animals enhance activation of the swim circuit. Conversely, social subordinates display a decreased activation of the swim circuit, but an enhanced activation of the escape circuit. In an effort to understand how social status mediates these effects, we constructed a neurocomputational model of the escape and swim circuits. The model replicates our findings and suggests that social status-related shift in circuit dynamics could be mediated by changes in the relative excitability of the escape and swim networks. Together, our results reveal that changes in the excitabilities of the Mauthner command neuron for escape and the inhibitory interneurons that regulate swimming provide a cellular mechanism for the nervous system to adapt to changes in social conditions by permitting the animal to select a socially appropriate behavioral response.SIGNIFICANCE STATEMENT Understanding how social factors influence nervous system function is of great importance. Using zebrafish as a model system, we demonstrate how social experience affects decision making to enable animals to produce socially appropriate behavior. Based on experimental evidence and computational modeling, we show that behavioral decisions reflect the interplay between competing neural circuits whose activation thresholds shift in accordance with social status. We demonstrate this through analysis of the behavior and neural circuit responses that drive escape and swim behaviors in fish. We show that socially subordinate animals favor escape over swimming, while socially dominants favor swimming over escape. We propose that these differences are mediated by shifts in relative circuit excitability.

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.

Spatial variation of waterborne Escherichia coli - implications for routine water quality monitoring.

Escherichia coli are often used as faecal indicator bacteria (FIB) to provide a measure of microbial pollution in recreational and shellfish harvesting waters. However, although model forecasts for predicting the concentrations of FIB in surface waters are becoming more robust, they suffer from an inconsistency in quantification methods and an understanding of the spatial variation of FIB within a water course. The aim of this study was to investigate the transverse spatial variation in E. coli numbers (as an indicator of faecal pollution) across the estuary of the River Conwy, UK. Water samples were collected from four transverse transects across the estuary. Spatial variation of E. coli was significantly different from one side of the river to the other, although was not correlated with depth or the physiochemical properties of the water. Subsequently, microbial water quality classifications on the two opposite banks suggested very different levels of pollution coming down the river. This work has shown that the side of the river that routine water monitoring samples are taken from can make a significant difference to the classification of microbial water quality. This has important implications for sampling strategies and the use of microbial source tracking (MST) techniques.

Measuring functional improvement after total knee arthroplasty requires both performance-based and patient-report assessments: a longitudinal analysis of outcomes.

The purpose was to explore the responsiveness of both patient-report and performance-based outcome measures to determine functional changes during the acute and long-term postoperative recovery after total knee arthroplasty (TKA). One hundred patients scheduled for unilateral TKA underwent testing preoperatively and at 1 and 12 months postoperatively using the Delaware Osteoarthritis Profile. All physical performance measures decreased initially after surgery then increased in the long term; however, the perceived function did not follow the same trend, and some showed an increase immediately after surgery. Patient-report measures were variable, with no to small response early, but had excellent long-term responsiveness that was twice as large as performance measures. Patient perception fails to capture the acute functional declines after TKA and may overstate the long-term functional improvement with surgery.

E. coli Is a Poor End-Product Criterion for Assessing the General Microbial Risk Posed From Consuming Norovirus Contaminated Shellfish.

The fecal indicator organism (FIO) Escherichia coli is frequently used as a general indicator of sewage contamination and for evaluating the success of shellfish cleaning (depuration) processes. To evaluate the robustness of this approach, the accumulation, retention, and depuration of non-pathogenic E. coli, pathogenic E. coli O157:H7 and norovirus GII (NoV GII) RNA were evaluated using a combination of culture-based (E. coli) and molecular methods (E. coli, NoV GII) after exposure of mussels (Mytilus edulis) to water contaminated with human feces. We simulated water contamination after a point-source release from a combined sewer overflow (CSO) where untreated wastewater is released directly into the coastal zone. All three microbiological indicators accumulated rapidly in the mussels, reaching close to maximum concentration within 3 h of exposure, demonstrating that short CSO discharges pose an immediate threat to shellfish harvesting areas. Depuration (72 h) in clean water proved partially successful at removing both pathogenic and non-pathogenic E. coli from shellfish tissue, but failed to eradicate NoV GII RNA. We conclude that current EU standards for evaluating microbiological risk in shellfish are inadequate for protecting consumers against exposure to human norovirus GII found in polluted marine waters.

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.

Use of Mytilus edulis biosentinels to investigate spatial patterns of norovirus and faecal indicator organism contamination around coastal sewage discharges.

Bivalve shellfish have the capacity to accumulate norovirus (NoV) from waters contaminated with human sewage. Consequently, shellfish represent a major vector for NoV entry into the human food chain, leading to gastrointestinal illness. Identification of areas suitable for the safe cultivation of shellfish requires an understanding of NoV behaviour upon discharge of municipal-derived sewage into coastal waters. This study exploited the potential of edible mussels (Mytilus edulis) to accumulate NoV and employed the ISO method for quantification of NoV within mussel digestive tissues. To evaluate the spatial spread of NoV from an offshore sewage discharge pipe, mesh cages of mussels were suspended from moorings deployed in a 9 km2 grid array around the outfall. Caged mussels were retrieved after 30 days and NoV (GI and GII), total coliforms and E. coli enumerated. The experimentally-derived levels of NoV GI and GII in mussels were similar with total NoV levels ranging from 7 × 101 to 1.6 × 104 genome copies g-1 shellfish digestive gland (ΣGI + GII). NoV spread from the outfall showed a distinct plume which matched very closely to predictions from the tidally-driven effluent dispersal model MIKE21. A contrasting spatial pattern was observed for coliforms (range 1.7 × 102 to 2.1 × 104 CFU 100 g-1 shellfish tissue) and E. coli (range 0-1.2 × 103 CFU 100 g-1 shellfish tissue). These data demonstrate that hydrodynamic models may help inform effective exclusion zones for bivalve harvesting, whilst coliform/E. coli concentrations do not accurately reflect viral dispersal in marine waters and contamination of shellfish by sewage-derived viral pathogens.

Spatial and temporal heterogeneity of bacteria across an intertidal shellfish bed: implications for regulatory monitoring of faecal indicator organisms.

Routine bacterial monitoring of shellfish beds using indicator species is a common global practice designed to prevent human consumption of contaminated shellfish products. However, current bacteriological monitoring procedures which focus on the quantification of faecal indicator organisms (FIOs) as a proxy for microbial pollution may not be representative of total bacterial contamination levels present in shellfish harvesting areas. The objective of this study was to critically assess the accuracy of current monitoring strategies by quantifying the spatial (lateral and longitudinal distance) and temporal (seasonality and tidal state) concentrations of FIOs (Escherichia coli and total coliforms) within a single intertidal commercially harvested shellfish bed. Spatial and temporal FIO dynamics, including the effects of tidal state and seasonality, were quantified in mussel flesh and sediment samples from a single intertidal mussel (Mytilus edulis) bed. Our results confirmed that FIO concentrations across a shellfish bed were heterogeneous over larger spatial and temporal scales, but showed no relation to the concentrations of autochthonous bacteria, such as Vibrio spp., or the physico-chemical parameters of the sediment. These results have important implications for both public health and the economic prosperity of the shellfish industry, and demonstrate the importance of accommodating both spatial and temporal fluctuations in routine bacteriological monitoring protocols. We conclude that current FIO monitoring procedures may not accurately represent levels of microbial contamination within shellfish harvesting areas and that more robust microbiological testing procedures need developing.

Sediment composition influences spatial variation in the abundance of human pathogen indicator bacteria within an estuarine environment.

Faecal contamination of estuarine and coastal waters can pose a risk to human health, particularly in areas used for shellfish production or recreation. Routine microbiological water quality testing highlights areas of faecal indicator bacteria (FIB) contamination within the water column, but fails to consider the abundance of FIB in sediments, which under certain hydrodynamic conditions can become resuspended. Sediments can enhance the survival of FIB in estuarine environments, but the influence of sediment composition on the ecology and abundance of FIB is poorly understood. To determine the relationship between sediment composition (grain size and organic matter) and the abundance of pathogen indicator bacteria (PIB), sediments were collected from four transverse transects of the Conwy estuary, UK. The abundance of culturable Escherichia coli, total coliforms, enterococci, Campylobacter, Salmonella and Vibrio spp. in sediments was determined in relation to sediment grain size, organic matter content, salinity, depth and temperature. Sediments that contained higher proportions of silt and/or clay and associated organic matter content showed significant positive correlations with the abundance of PIB. Furthermore, the abundance of each bacterial group was positively correlated with the presence of all other groups enumerated. Campylobacter spp. were not isolated from estuarine sediments. Comparisons of the number of culturable E. coli, total coliforms and Vibrio spp. in sediments and the water column revealed that their abundance was 281, 433 and 58-fold greater in sediments (colony forming units (CFU)/100g) when compared with the water column (CFU/100ml), respectively. These data provide important insights into sediment compositions that promote the abundance of PIB in estuarine environments, with important implications for the modelling and prediction of public health risk based on sediment resuspension and transport.