In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

Abstract

Optogenetic and pharmacogenetic techniques have been effective to analyze the role of neuronal activity in controlling oligodendroglia lineage cells in behaving juvenile and adult mice. This kind of studies is also of high interest during early postnatal (PN) development since important changes in oligodendroglia dynamics occur during the first two PN weeks. Yet, neuronal manipulation is difficult to implement at an early age because high-level, specific protein expression is less reliable in neonatal mice. Here, we describe a protocol allowing for an optogenetic stimulation of neurons in awake mouse pups with the purpose of investigating the effect of neuronal activity on oligodendroglia dynamics during early PN stages. Since GABAergic interneurons contact oligodendrocyte precursor cells (OPCs) through bona fide synapses and maintain a close relationship with these progenitors during cortical development, we used this relevant example of neuron-oligodendroglia interaction to implement a proof-of-principle optogenetic approach. First, we tested Nkx2.1-Cre and Parvalbumin (PV)-Cre lines to drive the expression of the photosensitive ion channel channelrhodopsin-2 (ChR2) in subpopulations of interneurons at different developmental stages. By using patch-clamp recordings and photostimulation of ChR2-positive interneurons in acute somatosensory cortical slices, we analyzed the level of functional expression of ChR2 in these neurons. We found that ChR2 expression was insufficient in PV-Cre mouse at PN day 10 (PN10) and that this channel needs to be expressed from embryonic stages (as in the Nkx2.1-Cre line) to allow for a reliable photoactivation in mouse pups. Then, we implemented a stereotaxic surgery to place a mini-optic fiber at the cortical surface in order to photostimulate ChR2-positive interneurons at PN10. In vivo field potentials were recorded in Layer V to verify that photostimulation reaches deep cortical layers. Finally, we analyzed the effect of the photostimulation on the layer V oligodendroglia population by conventional immunostainings. Neither the total density nor a proliferative fraction of OPCs were affected by increasing interneuron activity in vivo, complementing previous findings showing the lack of effect of GABAergic synaptic activity on OPC proliferation. The methodology described here should provide a framework for future investigation of the role of early cellular interactions during PN brain maturation.

Novel Tools and Investigative Approaches for the Study of Oligodendrocyte Precursor Cells (NG2-Glia) in CNS Development and Disease

“…Upon light activation on 10-days old pups, cortical OPC proliferation and density was analyzed. However, neither were affected by the increased interneuron activity, thus supporting previous findings indicating that postnatal GABAergic activity does not affect cortical oligodendroglia (Balia et al, 2017 ; Ortolani et al, 2018 ). Optogenetics also allows the control of gene expression via light exposure (Yamada et al, 2020 ) and modulation of gene expression in OPCs in vivo by shining light in a region-specific manner is an approach that could be used in the future.…”

“…Novel techniques of improving our understanding of OPC functions include optogenetic, intra cellular ion signaling activity and imaging (Friess et al, 2016 ; Ortolani et al, 2018 ; Zhang M. et al, 2019 ; Heredia et al, 2020 ; Marisca et al, 2020 ). Optogenetics involves the use of light to modulate cells expressing a light-sensitive ion channel, and has historically been used primarily to modulate neuronal activity (Lee et al, 2020 ).…”

“…Activation of ChR2 leads to the depolarization of the cell membrane which, in neurons, could trigger an action potential (Ernst et al, 2008 ). To study the importance of the transient early postnatal synaptic input from GABAergic interneurons to OPCs (Vélez-Fort et al, 2010 ), optogenetic methods have been used (Ortolani et al, 2018 ). Using Nkx2.1 -Cre and Parvalbumin Cre , ChR2 was expressed in a subpopulation of interneurons.…”

Myelination of parvalbumin interneurons shapes the function of cortical sensory inhibitory circuits

“…at the peak of FSI-OPC synaptic activity 8,14,27 , suggesting that besides inducing an aberrant myelination and axon morphology, the inactivation of γ2-mediated synapses of OPCs at an early postnatal period compromised FSI maturation. In normal conditions, moderate levels of PV mRNAs and protein expression in FSI at P10 increase during the following three postnatal weeks in the somatosensory cortex 25,27 . To assess potential FSI developmental impairments in γ2 f/f mice, we analyzed the density of PV cells at P10, P24, P30, and P120 ( Fig.…”

Glutamate versus GABA in neuron–oligodendroglia communication

“…Unlike expected, the inactivation of these synapses does not impact OPC proliferation and differentiation, but induces a moderate decrease in OPC density, suggesting a role of these synapses in OPC survival rather than in OL lineage cell development (Figure b; Balia et al, ). In line with this, the in vivo optogenetic stimulation of MGE‐derived interneurons in mouse pups does not modify the proliferation rate of OPCs (Ortolani, Manot‐Saillet, Orduz, Ortiz, & Angulo, ). Although these experiments are not specific of GABAergic synapses and are not necessarily correlated with the previous study, a stimulation of glutamatergic fibers does induce rapid changes in OPC prolifaration in normal (Gibson et al, ) and demyelinated conditions (Ortiz et al, ), suggesting that the activity of glutamatergic and GABAergic neurons may exert different effects or effects that can be modulated according to the receptors activated or the conditions.…”