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

To investigate the morphological features of chronic inflammatory demyelinating polyneuropathy (CIDP) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes.

Paranodal dissection in chronic inflammatory demyelinating polyneuropathy with anti-neurofascin-155 and anti-contactin-1 antibodies.

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.

Implications for White Matter Vulnerability to Anti-interleukin-6 Receptor Antibody Treatment.

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.

Cholinergic neural activation from the medial septal nucleus to hippocampus plays a crucial role in episodic memory as a regulating system for glutamatergic neural activation in the hippocampus. As a candidate regulating factor for acetylcholine synthesis in the medial septal nucleus, hippocampal cholinergic neurostimulating peptide (HCNP) was purified from the soluble fraction of young adult rat hippocampus. HCNP is released from its precursor protein (HCNP-pp), also referred to as phosphatidylethanolamine-binding protein 1. We recently reported that HCNP-pp conditional knockout (KO) mice, in which the HCNP-pp gene was knocked out at 3 months of age by tamoxifen injection, display no significant behavioral abnormalities, whereas HCNP-pp KO mice have a diminished cholinergic projection to CA1 and a decreased of theta activity in CA1. In this study, to address whether HCNP-pp reduction in early life is associated with behavioral changes, we evaluated the behavior of HCNP-pp KO mice in which HCNP-pp was downregulated from an early phase (postnatal days 14-28). As unexpected, HCNP-pp KO mice had no behavioral deficits. However, a significant positive correlation between HCNP-pp and gamma-aminobutyric acid A (GABA) receptor α3 subunit mRNA expression was found in individuals. This finding suggests involvement of HCNP-pp in regulating GABA receptor α3 gene expression.

Possible correlated variation of GABA receptor α3 expression with hippocampal cholinergic neurostimulating peptide precursor protein in the hippocampus.

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.

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.

Daisuke Kato

Department of Anatomy and Molecular Cell Biology

Nagoya University Graduate School of Medicine

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