Efficient Retrograde Monosynaptic Circuit Mapping Tool for Larval Zebrafish

Core Concepts

An optimized retrograde monosynaptic viral tracing system enables efficient mapping of neural circuits in the larval zebrafish brain.

Abstract

The authors developed an efficient and applicable retrograde monosynaptic viral tracing system using the EnvA-pseudotyped glycoprotein (G)-deleted rabies virus (RVdG[EnvA]) in larval zebrafish.

Key highlights:

• Transient co-expression of helper proteins TVA and rabies G in specific neurons via one-cell-stage microinjection of GAL4-UAS plasmids enabled RVdG[EnvA] to infect and spread from targeted starter cells.
• Iterative testing of virus strain, G protein type, G expression level, and rearing temperature identified an optimal combination of CVSdG trans-complemented with advanced expression of N2cG at 36°C, yielding up to 20 inputs per starter cell.
• The low cytotoxicity of the optimal conditions enabled viable labeling and calcium imaging of infected neurons up to 10 days post-infection, spanning larval ages commonly used for functional studies.
• A Cre-dependent transgenic reporter framework was developed to enable input cell-type-specific tracing and circuit reconstruction based on single-neuron morphology.
• Application of the method revealed the ipsilateral preference and subtype specificity of granule cell-to-Purkinje cell connections in the larval zebrafish cerebellum.

The authors' optimized viral tracing system provides an efficient tool for dissecting neural circuits in the larval zebrafish brain.

Stats

The tracing efficiency, measured by the convergence index (CI), was up to 20 for neurons under the optimal conditions of CVSdG trans-complemented with advanced expression of N2cG at 36°C.
The survival rate of larvae infected with CVSdG was significantly higher than those infected with SADdG at 36°C (75% ± 3% vs 52% ± 7%).
The lifetime of starter cells infected by CVSdG was longer than those infected by SADdG (10.0 ± 0 days vs 7.5 ± 0.48 days).

Quotes

"We discovered an optimal combinatory condition: CVSdG trans-complemented with advanced expression of N2cG at 36°C. The optimality lies in the high tracing efficiency and low cytotoxicity, embodied by a 20-fold increase in efficiency compared to previous studies in zebrafish and a long enough time window (final fish age up to 2 - 3 weeks old) for conducting functional studies in larval zebrafish."
"Consistently, in zebrafish larvae, we found that neurons infected with CVS-N2c displayed lower fluorescence intensity and longer survival times compared to those infected with SAD-B19."
"Importantly, in line with previous reports, we did not observe any noteworthy increase in mortality or behavioral abnormalities among zebrafish larvae exposed to elevated temperatures."

Key Insights Distilled From

An applicable and efficient retrograde monosynaptic circuit mapping tool for larval zebrafish

by Chen,T., Den... at www.biorxiv.org 07-02-2024

https://www.biorxiv.org/content/10.1101/2024.06.27.601104v1

Deeper Inquiries

How could the Cre-dependent tracing framework be further expanded to target specific neuronal subtypes or cell types beyond the current pan-neuronal labeling?

The Cre-dependent tracing framework can be expanded to target specific neuronal subtypes or cell types by incorporating cell type-specific promoters into the system. Currently, the framework utilizes the elavl3 promoter for pan-neuronal labeling, but by replacing this promoter with cell type-specific promoters, such as vglut2a for excitatory neurons or gad1b for inhibitory neurons, the tracing can be restricted to specific neuronal subtypes. This modification would allow for more precise circuit mapping and analysis by targeting distinct populations of neurons within the brain. Additionally, the incorporation of multiple Cre-dependent reporters with different fluorophores can enable the simultaneous visualization of multiple neuronal subtypes within the same circuit, providing a comprehensive view of the connectivity patterns in the brain.

What are the potential limitations or caveats of the viral tracing approach, and how could they be addressed to improve the reliability and interpretability of the circuit mapping results?

One potential limitation of the viral tracing approach is the possibility of non-specific labeling, such as the transneuronal spread to non-neuronal cells like glia. This can complicate the interpretation of circuit mapping results and may lead to inaccuracies in the connectivity patterns identified. To address this limitation, the viral tracing approach could be combined with cell type-specific genetic tools, such as the Cre-dependent tracing system described in the study. By using cell type-specific promoters and reporters, the tracing can be restricted to the desired neuronal populations, reducing the likelihood of non-specific labeling. Additionally, the optimization of viral strains and helper proteins, as demonstrated in the study, can improve the efficiency and specificity of the tracing, enhancing the reliability and interpretability of the circuit mapping results.

Given the insights into the cerebellar circuit wiring revealed by this study, how could the optimized viral tracing tool be leveraged to investigate the functional roles of different granule cell and Purkinje cell subtypes in zebrafish behavior and information processing?

The optimized viral tracing tool can be leveraged to investigate the functional roles of different granule cell (GC) and Purkinje cell (PC) subtypes in zebrafish behavior and information processing by combining the tracing with functional imaging techniques. By infecting specific GC and PC subtypes with the optimized viral tool and conducting calcium imaging or electrophysiological recordings, researchers can monitor the activity of these neurons in response to various stimuli or behavioral tasks. This approach can provide insights into the functional connectivity and information processing within the cerebellar circuit, shedding light on the roles of different neuronal subtypes in zebrafish behavior. Additionally, the combination of viral tracing with optogenetic manipulation techniques can allow for the precise control of neuronal activity, enabling the investigation of causal relationships between specific neuronal subtypes and behavioral outcomes.