Axonal and Dendritic Morphology of Excitatory Neurons in Layer 2/3 Mouse Barrel Cortex Imaged Through Whole-Brain Two-Photon Tomography and Registered to a Digital Brain Atlas.

Yanqi Liu,Georgios Foustoukos,Sylvain Crochet,Carl C.H Petersen

doi:10.3389/fnana.2021.791015

Yanqi Liu, Georgios Foustoukos + Show 2 more

Open Access

https://doi.org/10.3389/fnana.2021.791015

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Abstract

Communication between cortical areas contributes importantly to sensory perception and cognition. On the millisecond time scale, information is signaled from one brain area to another by action potentials propagating across long-range axonal arborizations. Here, we develop and test methodology for imaging and annotating the brain-wide axonal arborizations of individual excitatory layer 2/3 neurons in mouse barrel cortex through single-cell electroporation and two-photon serial section tomography followed by registration to a digital brain atlas. Each neuron had an extensive local axon within the barrel cortex. In addition, individual neurons innervated subsets of secondary somatosensory cortex; primary somatosensory cortex for upper limb, trunk, and lower limb; primary and secondary motor cortex; visual and auditory cortical regions; dorsolateral striatum; and various fiber bundles. In the future, it will be important to assess if the diversity of axonal projections across individual layer 2/3 mouse barrel cortex neurons is accompanied by functional differences in their activity patterns.

Highlights

On the millisecond timescale, neurons communicate primarily by releasing neurotransmitters from presynaptic specializations along their axons in response to action potential firing, with the increased concentration of neurotransmitters acting to open ligand-gated ion channels largely concentrated in postsynaptic specializations on dendrites
We performed two-photon guided in vivo ‘‘shadow’’ electroporation to label single neurons in layer 2/3 of mouse barrel cortex by expression of GFP (Judkewitz et al, 2009)
Through two-photon tomographic imaging and threedimensional neuron reconstruction (Han et al, 2018) in relation to a digital mouse brain atlas, we quantified long-range projection regions among the 10 reconstructed cells finding a high degree of diversity

Summary

Introduction

Neurons communicate primarily by releasing neurotransmitters from presynaptic specializations along their axons in response to action potential firing, with the increased concentration of neurotransmitters acting to open ligand-gated ion channels largely concentrated in postsynaptic specializations on dendrites. In this study, we sampled layer 2/3 mouse barrel cortex neurons without pre-labeling of their long-range projections, and we made three important methodological advances over our previous work towards quantitatively studying their anatomy: (i) we imaged entire mouse brains using two-photon serial section tomography; (ii) we registered our data to a standardized digital atlas of the mouse brain; and (iii) we quantified axonal length in the context of brain areas annotated in the digital atlas Through these technical advances, we have begun to further characterize the diversity of the axonal projections of individual layer 2/3 neurons in the mouse barrel cortex, adding to the important body of previous knowledge about the single-cell anatomy of excitatory projection neurons in the superficial layers of rodent primary whisker somatosensory cortex (Feldmeyer et al, 2006; Lübke and Feldmeyer, 2007; Yamashita et al, 2018; Egger et al, 2020; Peng et al, 2021; Staiger and Petersen, 2021). Immunohistochemical labeling of processed tissue pointed to important technical limitations indicating that our methodology revealed incomplete axonal arborizations

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