An inhalation anaesthesia approach for neonatal mice allowing streamlined stereotactic injection in the brain

Hinze Ho,Adam Fowle,Marisa Coetzee,Ingo H Greger,Jake F Watson

doi:10.1016/j.jneumeth.2020.108824

Hinze Ho, Adam Fowle + Show 3 more

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https://doi.org/10.1016/j.jneumeth.2020.108824

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Abstract

BackgroundInvestigating brain function requires tools and techniques to visualise, modify and manipulate neuronal tissue. One powerful and popular method is intracerebral injection of customised viruses, allowing expression of exogenous transgenes. This technique is a standard procedure for adult mice, and is used by laboratories worldwide. Use of neonatal animals in scientific research allows investigation of developing tissues and enables long-term study of cell populations. However, procedures on neonatal mice are more challenging, due to the lack of reliable methods and apparatus for anaesthesia of these animals. New methodHere, we report an inhalation-based protocol for anaesthesia of neonatal (P0−2) mice and present a custom 3D-printed apparatus for maintenance of anaesthesia during surgical procedures. Our optimised method of anaesthesia enables a rapid method of stereotactic injection in neonatal mice for transduction of brain tissue. Results and comparison with existing methodsThis approach significantly enhances animal welfare and facilitates wider and simpler use of neonatal rodents in scientific research. We demonstrate this procedure for targeted labelling of specific brain regions, and in vivo modification of tissue prior to organotypic culture. ConclusionsOur protocol for reliable delivery of inhalational anaesthetics can be readily adopted by any laboratory and will enable safer use of neonatal rodents across a diverse spectrum of scientific disciplines. Application to stereotactic injections allows a rapid and efficient method for modification of brain tissue.

Highlights

Understanding the mechanisms of brain function requires approaches that reveal the structure and activity of neuronal networks
We demonstrate this procedure for targeted labelling of specific brain regions, and in vivo modification of tissue prior to organotypic culture
We aimed to develop a robust and reliable technique for anaesthetising neonatal mice, as an alternative to hypothermia

Summary

Introduction

Understanding the mechanisms of brain function requires approaches that reveal the structure and activity of neuronal networks. Recombinant DNA technology has enabled development of powerful genetically encoded tools for visualisation of cell populations, editing of genomic DNA, and tracing of neuronal circuits. These tools can be delivered in repurposed viruses into specific brain areas of adult rodents using intracerebral stereotactic injections (Cetin et al, 2006; Sun and Schaffer, 2018). This widely performed technique involves surgically opening the skull before injection, requiring approximately 40 mins per animal (Cetin et al, 2006), but is highly efficient and provides reliable neuronal transduction in adult animals. In accordance with 3Rs principles, this approach is being restricted (Herrmann and Flecknell, 2019)

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