evaluation-manipulation-neural-activity-using-two-photon-holographic

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

Nagoya University Graduate School of Medicine

National Institutes of Natural Sciences

Kobe University

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood-brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

Dual microglia effects on blood brain barrier permeability induced by systemic inflammation.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor AR are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

Negative feedback control of neuronal activity by microglia.

Maternal infection or inflammation causes abnormalities in brain development associated with subsequent cognitive impairment and in an increased susceptibility to schizophrenia and autism spectrum disorders. Maternal immune activation (MIA) and increases in serum cytokine levels mediates this association via effects on the fetal brain, and microglia can respond to maternal immune status, but consensus on how microglia may respond is lacking and no-one has yet examined if microglial process motility is impaired. In this study we investigated how MIA induced at two different gestational ages affected microglial properties at different developmental stages. Immune activation in mid-pregnancy increased IL-6 expression in embryonic microglia, but failed to cause any marked changes in morphology either at E18 or postnatally. In contrast MIA, particularly when induced earlier (at E12), caused sustained alterations in the patterns of microglial process motility and behavioral deficits. Our research has identified an important microglial property that is altered by MIA and which may contribute to the underlying pathophysiological mechanisms linking maternal immune status to subsequent risks for cognitive disease.

Maternal immune activation induces sustained changes in fetal microglia motility.

Oligodendrocytes, which form the myelin sheaths that insulate axons, regulate conduction velocity. Myelinated axons make up the brain's white matter and contribute to the efficiency of information processing by regulating the timing of neural activity. Traditionally, it has been thought that myelin is a static, inactive insulator around the axon. However, recent studies in humans using magnetic resonance imaging have shown that structural changes in the white matter occur during learning and training, suggesting that 1) white matter change depends on neural activity and 2) activity-dependent changes in white matter are essential for learning and behavior. Furthermore, suppression of oligodendrocytes and their progenitor cells leads to deficits in motor learning and remote fear memory consolidation, suggesting a causal relationship between glial function and the learning process. However, for technical reasons, it remains unclear how myelin-generating glia modulate neural circuitry and what underlying mechanisms they employ to affect learning and behavior. Recent advances in optical and genetic techniques have helped elucidate this mechanism. In this review, we highlight evidence that neural activities regulated by myelin plasticity play a pivotal role in learning and behavior and provide further insight into possible therapeutic targets for treating diseases accompanied by myelin impairment.

Myelin plasticity modulates neural circuitry required for learning and behavior.

Sustained neuropathic pain from injury or inflammation remains a major burden for society. Rodent pain models have informed some cellular mechanisms increasing neuronal excitability within the spinal cord and primary somatosensory cortex (S1), but how activity patterns within these circuits change during pain remains unclear. We have applied multiphoton in vivo imaging and holographic stimulation to examine single S1 neuron activity patterns and connectivity during sustained pain. Following pain induction, there is an increase in synchronized neuronal activity and connectivity within S1, indicating the formation of pain circuits. Artificially increasing neuronal activity and synchrony using DREADDs reduced pain thresholds. The expression of N-type voltage-dependent Ca channel subunits in S1 was increased after pain induction, and locally blocking these channels reduced both the synchrony and allodynia associated with inflammatory pain. Targeting these S1 pain circuits, via inhibiting N-type Ca channels or other approaches, may provide ways to reduce inflammatory pain.

Pain induces stable, active microcircuits in the somatosensory cortex that provide a therapeutic target.

The central nervous system (CNS) is an immune-privileged area. The blood-brain barrier (BBB) is thought to separate the CNS from any systemic inflammatory states to maintain homeostasis within this specialized, vulnerable organ. However, accumulating studies have challenged this concept by demonstrating systemic inflammatory effects on brain. Moreover, the coronavirus disease pandemic caused by severe acute respiratory syndrome coronavirus 2 in 2019 has rapidly evoked attention toward the BBB as the systemic-CNS immunological interface. In this review, we focus on microglia, the sole immune cells in the CNS, and briefly introduce our new findings regarding microglial BBB regulation in systemic inflammation. With a close eye on associated literature, we carefully rethink the traditional immune system in the CNS and suggest a new possible mechanism of systemic-CNS immune cell interaction, while an understanding of the BBB develops.

[Microglial Regulation of Blood Brain Barrier, the Neuro-Immunological Interface].

Holographic structured illumination combined with optogenetics enables patterned stimulation of neurons and glial cells in an intact living brain. Moreover, in vivo functional imaging of cellular activity with recent advanced microscope technologies allows for visualization of the cellular responses during learning, emotion and cognition. Integrating these techniques can be used to verify the link between cell function and behavior output. However, there are technical limitations to stimulate multiple cells with high spatial and temporal resolution with available techniques of optogenetic stimulation. Here, we summarized a two-photon microscope combined with holographic system to stimulate multiple cells with high spatial and temporal resolution for living mice and their biological application.

Holographic microscope and its biological application.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the cytosolic Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.

A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy.

Neonicotinoid pesticides are a class of insecticides that reportedly have harmful effects on bees and dragonflies, causing a reduction in their numbers. Neonicotinoids act as neuroreceptor modulators, and some studies have reported their association with neurodevelopmental disorders. However, the precise effect of neonicotinoids on the central nervous system has not yet been identified. Herein, we conducted in vivo Ca imaging using a two-photon microscope to detect the abnormal activity of neuronal circuits in the brain after neonicotinoid application. The oral administration of acetamiprid (ACE) (20 mg/kg body weight (BW) in mature mice with a quantity less than the no-observed-adverse-effect level (NOAEL) and a tenth or half of the median lethal dose (LD) of nicotine (0.33 or 1.65 mg/kg BW, respectively), as a typical nicotinic acetylcholine receptor (nAChR) agonist, increased anxiety-like behavior associated with altered activities of the neuronal population in the somatosensory cortex. Furthermore, we detected ACE and its metabolites in the brain, 1 h after ACE administration. The results suggested that in vivo Ca imaging using a two-photon microscope enabled the highly sensitive detection of neurotoxicant-mediated brain disturbance of nerves.

Article	Year
Dual microglia effects on blood brain barrier permeability induced by systemic inflammation. Nature communications\| PubMed ID: 31862977	2019
Negative feedback control of neuronal activity by microglia. Nature\| PubMed ID: 32999463	2020
Maternal immune activation induces sustained changes in fetal microglia motility. Scientific reports\| PubMed ID: 33288794	2020
Myelin plasticity modulates neural circuitry required for learning and behavior. Neuroscience research\| PubMed ID: 33417972	2021
Pain induces stable, active microcircuits in the somatosensory cortex that provide a therapeutic target. Science advances\| PubMed ID: 33741588	2021
[Microglial Regulation of Blood Brain Barrier, the Neuro-Immunological Interface]. Brain and nerve = Shinkei kenkyu no shinpo\| PubMed ID: 34376598	2021
Holographic microscope and its biological application. Neuroscience research\| PubMed ID: 34740727	2022
A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy. The Journal of clinical investigation\| PubMed ID: 35290243	2022
Ca imaging with two-photon microscopy to detect the disruption of brain function in mice administered neonicotinoid insecticides. Scientific reports\| PubMed ID: 35332220	2022
Lighting up cosmic neuronal networks with transformative in vivo calcium imaging. Neuroscience research\| PubMed ID: 35460729	2022

Hiroaki Wake

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