in-vivo-two-photon-imaging-of-cortical-neurons-in-neonatal-mice

<em>In Vivo</em> Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

in-vivo-visualization-spontaneous-activity-neonatal-mouse-sensory

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

Kumamoto University

National Institute of Genetics

SOKENDAI (The Graduate University for Advanced Studies)

Proper neuronal circuit function relies on precise dendritic projection, which is established through activity-dependent refinement during early postnatal development. Here we revealed dynamics of dendritic refinement in the mammalian brain by conducting long-term imaging of the neonatal mouse barrel cortex. By "retrospective" analyses, we identified "prospective" barrel-edge spiny stellate (SS) neurons in early neonates, which had an apical dendrite and primitive basal dendrites (BDs). These neurons retracted the apical dendrite gradually and established strong BD orientation bias through continuous "dendritic tree" turnover. A greater chance of survival was given to BD trees emerged in the barrel-center side, where thalamocortical axons (TCAs) cluster. When the spatial bias of TCA inputs to SS neurons was lost, BD tree turnover was suppressed, and most BD trees became stable and elaborated mildly. Thus, barrel-edge SS neurons could establish the characteristic BD projection pattern through differential dynamics of dendritic trees induced by spatially biased inputs.

Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates.

HSCs can be separated based on high or low mitochondrial mass. Higher mitochondrial mass is associated with quiescence and greater reconstitution capacity of HSCs.

High mitochondrial mass is associated with reconstitution capacity and quiescence of hematopoietic stem cells.

Spatially-organized spontaneous activity is a characteristic feature of developing mammalian sensory systems. However, the transitions of spontaneous-activity spatial organization during development and related mechanisms remain largely unknown. We reported previously that layer 4 (L4) glutamatergic neurons in the mouse barrel cortex exhibit spontaneous activity with a patchwork-type pattern at postnatal day (P)5, which is during barrel formation. In the current work, we revealed that spontaneous activity in mouse barrel-cortex L4 glutamatergic neurons exhibits at least three phases during the first two weeks of postnatal development. Phase I activity has a patchwork-type pattern and is observed not only at P5, but also P1, before barrel formation. Phase II is found at P9, by which time barrel formation is completed, and exhibits broadly synchronized activity across barrel borders. Phase III emerges around P11 when L4-neuron activity is desynchronized. The Phase I activity, but not Phase II or III activity, is blocked by thalamic inhibition, demonstrating that the Phase I to II transition is associated with loss of thalamic dependency. Dominant-negative (DN)-Rac1 expression in L4 neurons hampers the Phase II to III transition. It also suppresses developmental increases in spine density and excitatory synapses of L4 neurons in the second postnatal week, suggesting that Rac1-mediated synapse maturation could underlie the Phase II to III transition. Our findings revealed the presence of distinct mechanisms for Phase I to II and Phase II to III transition. They also highlighted the role of a small GTPase in the developmental desynchronization of cortical spontaneous activity. Developing neocortex exhibits spatially-organized spontaneous activity, which plays a critical role in cortical circuit development. The features of spontaneous-activity spatial organization and the mechanisms underlying its changes during development remain largely unknown. In the present study, using two-photon  imaging, we revealed three phases (Phases I, II, and III) of spontaneous activity in barrel-cortex layer 4 (L4) glutamatergic neurons during the first two postnatal weeks. We also demonstrated the presence of distinct mechanisms underlying phase transitions. Phase I to II shift arose from the switch in the L4-neuron driving source, and Phase II to III transition relied on L4-neuron Rac1 activity. These results provide new insights into the principles of developmental transitions of neocortical spontaneous-activity spatial patterns.

Developmental Phase Transitions in Spatial Organization of Spontaneous Activity in Postnatal Barrel Cortex Layer 4.

The cerebral cortex has complex yet perfectly wired neuronal circuits that are important for high-level brain functions such as perception and cognition. The rodent's somatosensory system is widely used for understanding the mechanisms of circuit formation during early developmental periods. In this review, we summarize the developmental processes of circuit formation in layer 4 of the somatosensory cortex, and we describe the molecules involved in layer 4 circuit formation and neuronal activity-dependent mechanisms of circuit formation. We also introduce the dynamic mechanisms of circuit formation in layer 4 revealed by intravital two-photon imaging technologies, which include time-lapse imaging of neuronal morphology and calcium imaging of neuronal activity in newborn mice.

Elucidating mechanisms of neuronal circuit formation in layer 4 of the somatosensory cortex via intravital imaging.

Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that NMDA receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 (), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knock-out (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex. Precise wiring of the thalamocortical circuits is necessary for proper sensory information processing, and thalamus-derived correlated spontaneous activity is important for thalamocortical circuit formation. The molecular mechanisms involved in the correlated activity transfer from the thalamus to the neocortex are largely unknown.  two-photon calcium imaging of the neonatal barrel cortex revealed that correlated spontaneous activity between layer four neurons is reduced by mosaic knock-out (KO) of the NMDA receptor (NMDAR) obligatory subunit GluN1. Our results suggest that the function of NMDARs in layer four neurons is necessary for the communication between presynaptic and postsynaptic partners during thalamocortical circuit formation.

NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex.

In most dicotyledonous plants, leaf pavement cells exhibit complex jigsaw puzzle-like cell morphogenesis during leaf expansion. Although detailed molecular biological information and mathematical modeling of this jigsaw puzzle-like cell morphogenesis are now available, a full understanding of this process remains elusive. Recent reports have highlighted the importance of three-dimensional (3D) structures (i.e., anticlinal and periclinal cell wall) in understanding the mechanical models that describe this morphogenetic process. We believe that it is important to acquire 3D shapes of pavement cells over time, i.e., acquire and analyze four-dimensional (4D) information when studying the relationship between mechanical modeling and simulations and the actual cell shape. In this report, we have developed a framework to capture and analyze 4D morphological information of  cotyledon pavement cells by using both direct water immersion observations and computational image analyses, including segmentation, surface modeling, virtual reality and morphometry. The 4D cell models allowed us to perform time-lapse 3D morphometrical analysis, providing detailed quantitative information about changes in cell growth rate and shape, with cellular complexity observed to increase during cell growth. The framework should enable analysis of various phenotypes (e.g., mutants) in greater detail, especially in the 3D deformation of the cotyledon surface, and evaluation of theoretical models that describe pavement cell morphogenesis using computational simulations. Additionally, our accurate and high-throughput acquisition of growing cell structures should be suitable for use in generating  model cell structures.

Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of  cotyledon pavement cells.

Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.

Live imaging of avian epiblast and anterior mesendoderm grafting reveals the complexity of cell dynamics during early brain development.

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells.

The cerebral cortex comprises a complex and exquisite network of neuronal circuits that is formed during development. To explore the molecular mechanisms involved in cortical circuit formation, the tactile somatosensory pathway that connects the whiskers and cortex of rodents is a useful model. Here, we analyzed the roles of Ras GTPase-activating proteins (RasGAPs) in the circuit formation in the somatosensory cortex layer 4 (L4). We suppressed the function of RasGAPs in L4 neurons using Supernova RNAi, a plasmid vector-based sparse cell gene knockdown (KD) system. The results showed disrupted dendritic pattern formation of L4 spiny stellate neurons on the barrel edge by RasGAP KD. Furthermore, the number of presynaptic boutons on L4 neurons was reduced by RasGAP KD. These results demonstrate the essential roles of RasGAPs in circuit formation in the cerebral cortex and imply that developmental changes in dendrites and synapses in RasGAP KD neurons may be related to cognitive disabilities in RasGAP-deficient individuals, such as patients with neurofibromatosis type 1.

Ras GTPase-activating proteins control neuronal circuit development in barrel cortex layer 4.

Editorial:  investigations on neurological disorders: From traditional approaches to forefront technologies.

In vivo calcium imaging is essential to elucidate unique synchronous activities observed in the developing brain. Here, we present a protocol to image and analyze activity patterns in neonatal mouse neocortex in a single-cell level. We describe steps for in utero electroporation, cranial window surgery, two-photon imaging, and activity correlation analysis. This protocol facilitates the understanding of neuronal activities and activity-dependent circuit formation during development. For complete details on the use and execution of this protocol, please refer to Mizuno et al. (2014), Mizuno et al. (2018a), and Mizuno et al. (2018b)..

In vivo two-photon calcium imaging of cortical neurons in neonatal mice.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon  time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8 h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.

ArticleTotal : 2	Year
Publication title	2018
Publication title	2023

Article	Year
Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates. Nature communications\| PubMed ID: 30082783	2018
High mitochondrial mass is associated with reconstitution capacity and quiescence of hematopoietic stem cells. Blood advances\| PubMed ID: 31387881	2019
Developmental Phase Transitions in Spatial Organization of Spontaneous Activity in Postnatal Barrel Cortex Layer 4. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 32887743	2020
Elucidating mechanisms of neuronal circuit formation in layer 4 of the somatosensory cortex via intravital imaging. Neuroscience research\| PubMed ID: 33309867	2021
NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 33372060	2021
Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of cotyledon pavement cells. Plant biotechnology (Tokyo, Japan)\| PubMed ID: 33850430	2020
Live imaging of avian epiblast and anterior mesendoderm grafting reveals the complexity of cell dynamics during early brain development. Development (Cambridge, England)\| PubMed ID: 35132990	2022
Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells. Communications biology\| PubMed ID: 35918480	2022
Ras GTPase-activating proteins control neuronal circuit development in barrel cortex layer 4. Frontiers in neuroscience\| PubMed ID: 36188467	2022
Editorial: investigations on neurological disorders: From traditional approaches to forefront technologies. Frontiers in neuroscience\| PubMed ID: 36330344	2022
In vivo two-photon calcium imaging of cortical neurons in neonatal mice. STAR protocols\| PubMed ID: 37119143	2023
Short-Term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by Imaging with Improved Spatiotemporal Resolution. eNeuro\| PubMed ID: 37890991	2023

Hidenobu Mizuno

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