Two-photon GCaMP6f imaging of infrared neural stimulation evoked calcium signals in mouse cortical neurons in vivo

Attila Kaszas,Balázs Rózsa,Ivo Vanzetta,Rodney O'Connor,Andrea Slézia,Alexandra Bojdán,David Moreau,Balázs Hangya,Gergely Szalay

doi:10.1038/s41598-021-89163-x

Abstract

Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue. Very few studies have explored its use in the brain, as most investigations have focused on sensory or motor nerve stimulation. Using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f, here we show that the application of infrared neural stimulation induces intracellular calcium signals in Layer 2/3 neurons in mouse cortex in vivo. The number of neurons exhibiting infrared-induced calcium response as well as the amplitude of those signals are shown to be both increasing with the energy density applied. By studying as well the spatial extent of the stimulation, we show that reproducibility of the stimulation is achieved mainly in the central part of the infrared beam path. Stimulating in vivo at such a degree of precision and without any exogenous chromophores enables multiple applications, from mapping the brain’s connectome to applications in systems neuroscience and the development of new therapeutic tools for investigating the pathological brain.

Highlights

Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue
When the optical axis was perpendicular to the cortical surface, the best approach angle we could achieve with a bare fiber was about 26° in respect to the optical axis, which was impractical because the fiber adhered to either the microscope objective or to the cortex, hindering precise positioning
We showed that two-photon calcium imaging of mice expressing GCaMP6f in neurons is a useful tool to assess infrared-induced calcium signals in cortical layer 2/3 neurons in vivo

Summary

Introduction

Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue. An increasingly popular means for the optical stimulation of neurons is optogenetics[3] This method is less invasive, artefact free (at least in normal energy ranges) and has the ability to reach high spatial selectivity when used in optimized conditions; it requires the genetic manipulation of neurons in order for them to express light sensitive opsin-based ion channels. This is usually achieved through viral transfections, which—despite their good controllability and the advantage of offering cell type specificity through the use of a promoter—, strongly limits applicability in humans. Deep neuronal tissue infrared modulation was recently shown in rat neocortex and hippocampus, through the use of an implantable silicon microdevice enabling infrared delivery with an embedded waveguide and neuronal activity measurement with platinum microelectrodes[35]

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