Astrocytic modulation of population encoding in mouse visual cortex via GABA transporter 3 revealed by multiplexed CRISPR/Cas9 gene editing
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Curated by eLife
eLife Assessment
In this manuscript, Park et al. developed a multiplexed CRISPR construct to genetically ablate the GABA transporter GAT3 in the mouse visual cortex, with effects on population-level neuronal activity. This work is important, as it sheds light on how GAT3 controls the processing of visual information. The findings are compelling, leveraging state-of-the-art gene CRISPR/Cas9, in vivo two-photon laser scanning microscopy, and advanced statistical modeling.
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Abstract
Summary
Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.
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eLife Assessment
In this manuscript, Park et al. developed a multiplexed CRISPR construct to genetically ablate the GABA transporter GAT3 in the mouse visual cortex, with effects on population-level neuronal activity. This work is important, as it sheds light on how GAT3 controls the processing of visual information. The findings are compelling, leveraging state-of-the-art gene CRISPR/Cas9, in vivo two-photon laser scanning microscopy, and advanced statistical modeling.
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Reviewer #1 (Public review):
Summary:
The authors have investigated the role of GAT3 in the visual system. First, they have developed a CRISPR/Cas9-based approach to locally knock out this transporter in the visual cortex. They then demonstrated electrophysiologically that this manipulation increases inhibitory synaptic input into layer 2/3 pyramidal cells. They further examined the functional consequences by imaging neuronal activity in the visual cortex in vivo. They found that the absence of GAT3 leads to reduced spontaneous neuronal activity and attenuated neuronal responses and reliability to visual stimuli, but without an effect on orientation selectivity. Further analysis of this data suggests that Gat3 removal leads to less coordinated activity between individual neurons and in population activity patterns, thereby impairing …
Reviewer #1 (Public review):
Summary:
The authors have investigated the role of GAT3 in the visual system. First, they have developed a CRISPR/Cas9-based approach to locally knock out this transporter in the visual cortex. They then demonstrated electrophysiologically that this manipulation increases inhibitory synaptic input into layer 2/3 pyramidal cells. They further examined the functional consequences by imaging neuronal activity in the visual cortex in vivo. They found that the absence of GAT3 leads to reduced spontaneous neuronal activity and attenuated neuronal responses and reliability to visual stimuli, but without an effect on orientation selectivity. Further analysis of this data suggests that Gat3 removal leads to less coordinated activity between individual neurons and in population activity patterns, thereby impairing information encoding. Overall, this is an elegant and technically advanced study that demonstrates a new and important role of GAT3 in controlling the processing of visual information.
Strengths:
(1) Development of a new approach for a local knockout (GAT3).
(2) Important and novel insights into visual system function and its dependence on GAT3.
(3) Plausible cellular mechanism.
Weaknesses:
No major weaknesses were identified by this reviewer.
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Reviewer #2 (Public review):
Summary:
Park et al. have made a tool for spatiotemporally restricted knockout of the astrocytic GABA transporter GAT3, leveraging CRISPR/Cas9 and viral transduction in adult mice, and evaluated the effects of GAT3 on neural encoding of visual stimulation.
Strengths:
This concise manuscript leverages state-of-the-art gene CRISPR/Cas9 technology for knocking out astrocytic genes. This has only to a small degree been performed previously in astrocytes, and it represents an important development in the field. Moreover, the authors utilize in vivo two-photon imaging of neural responses to visual stimuli as a readout of neural activity, in addition to validating their data with ex vivo electrophysiology. Lastly, they use advanced statistical modeling to analyze the impact of GAT3 knockout. Overall, the study …
Reviewer #2 (Public review):
Summary:
Park et al. have made a tool for spatiotemporally restricted knockout of the astrocytic GABA transporter GAT3, leveraging CRISPR/Cas9 and viral transduction in adult mice, and evaluated the effects of GAT3 on neural encoding of visual stimulation.
Strengths:
This concise manuscript leverages state-of-the-art gene CRISPR/Cas9 technology for knocking out astrocytic genes. This has only to a small degree been performed previously in astrocytes, and it represents an important development in the field. Moreover, the authors utilize in vivo two-photon imaging of neural responses to visual stimuli as a readout of neural activity, in addition to validating their data with ex vivo electrophysiology. Lastly, they use advanced statistical modeling to analyze the impact of GAT3 knockout. Overall, the study comes across as rigorous and convincing.
Weaknesses:
Adding the following experiments would potentially have strengthened the conclusions and helped with interpreting the findings:
(1) Neural activity is quite profoundly influenced by GAT3 knockout. Corroborating these relatively large changes to neural activity with in vivo electrophysiology of some sort as an additional readout would have strengthened the conclusions.
(2) Given the quite large effects on neural coding in visual cortex assessed på jRGECO imaging, it would have been interesting if the mouse groups could have been subjected to behavioral testing, assessing the visual system.
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