Sleep deprivation impairs Ca2+ expression in the hippocampus: ionic imaging analysis for cognitive deficiency with TOF-SIMS

Sleep deprivation causes cognitive dysfunction in which impaired neuronal plasticity in hippocampus may underlie the molecular mechanisms of this deficiency. Considering calcium-mediated NMDA receptor subunit 1 (NMDAR1) and neuronal nitric oxide synthase (nNOS) activation plays an important role in the regulation of neuronal plasticity, the present study is aimed to determine whether total sleep deprivation (TSD) would impair calcium expression, together with injury of the neuronal plasticity in hippocampus. Adult rats subjected to TSD were processed for time-of-flight secondary ion mass spectrometry, NMDAR1 immunohistochemistry, nNOS biochemical assay, cytochrome oxidase histochemistry, and the Morris water maze learning test to detect ionic, neurochemical, bioenergetic as well as behavioral changes of neuronal plasticity, respectively. Results indicated that in normal rats, strong calcium signaling along with intense NMDAR1/nNOS expression were observed in hippocampal regions. Enhanced calcium imaging and neurochemical expressions corresponded well with strong bioenergetic activity and good performance of behavioral testing. However, following TSD, both calcium intensity and NMDAR1/nNOS expressions were significantly decreased. Behavioral testing also showed poor responses after TSD. As proper calcium expression is essential for maintaining hippocampal neuronal plasticity, impaired calcium expression would depress downstream NMDAR1-mediated nNOS activation, which might contribute to the initiation or development of TSD-related cognitive deficiency.     

Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.     

Optical recording of brain functions based on voltage-sensitive dyes

To better understand the spatial distribution of brain functions, we need to monitor and analyze neuronal activities. Electrophysiological technique has provided an important method for the exploration of some neural circuits. However, this method cannot simultaneously detect the activities of nerve cell groups.Therefore, methods that can monitor the spatial distribution of neuronal population activity are demanded to explore brain functions. Voltage-sensitive dyes(VSDs) shift their absorption or emission optical signals in response to different membrane potentials, allowing assessing the global electrical state of neurons. Optical recording technique coupled with VSDs is a promising method to monitor the brain functions by detecting optical signal changes. This review focuses on the fast and slow responses of VSDs to membrane potential changes and optical recordings utilized in the central nervous system. In this review, we attempt to show how VSDs and optical recordings can be used to obtain brain functional monitoring at high spatial and temporal resolution. Understanding of brain functions will not only greatly improve the cognition of information transmission of complex neural network, but also provide new methods of treating brain diseases such as Parkinson's and Alzheimer's diseases.     

Analysis of glutamate in striatal microdialysates using capillary electrophoresis and laser-induced fluorescence detection

High-performance liquid chromatography (HPLC) with electrochemical detection has been used routinely to analyse the neurochemical constituents of brain microdialysates. However, conventional HPLC analysis requires large injection volumes and hence lengthy dialysis sampling times. Capillary electrophoresis (CE) is a rapid high-resolution separation technique with the ability to routinely handle very small sample volumes. If CE is coupled to a high-sensitivity detection system, such as laser-induced fluorescence (LIF), it becomes a powerful and rapid separation technique for the analysis of small-volume microdialysis samples.

These preliminary studies report reduced separation times for the excitatory amino acid glutamate, prederivatised with naphthalene 2,3-dialdehyde, and demonstrate its detection within small-volume brain microdialysis samples. The limit of detection for this system was 10⁻⁸ M.

Characterisation of striatal microdialysis samples comprised infusions of Ca²⁺-free artificial cerebrospinal fluid (aCSF) and Tetrodotoxin (TTx) (10 mM) to demonstrate that the detected transmitter is of neuronal origin and released in a calcium-dependent manner.

Removal of calcium from aCSF resulted in a decrease in glutamate in dialysis samples. Glutamate release significantly decreased (p<0.05) to ca. 40% of preinfusion control levels after 60 min and this level was maintained throughout the sampling period. These data suggest that glutamate release is, to some degree, a calcium-dependent process. TTx infusion (10 μM) produced a significant (p<0.05) reduction in glutamate release to ca. 10% of preinfusion levels. It would therefore appear that glutamate release is dependent on neuronal activity. In summary, we have demonstrated the establishment of CE-LIF and microdialysis for the measurement of glutamate.     

Ring-shaped neuronal networks: a platform to study persistent activity

Persistent activity in the brain is involved in working memory and motor planning. The ability of the brain to hold information 'online' long after an initiating stimulus is a hallmark of brain areas such as the prefrontal cortex. Recurrent network loops such as the thalamocortical loop and reciprocal loops in the cortex are potential substrates that can support such activity. However, native brain circuitry makes it difficult to study mechanisms underlying such persistent activity. Here we propose a platform to study synaptic mechanisms of such persistent activity by constraining neuronal networks to a recurrent loop like geometry. Using a polymer stamping technique, adhesive proteins are transferred onto glass substrates in a precise ring shape. Primary rat hippocampal cultures were capable of forming ring-shaped networks containing 40-60 neurons. Calcium imaging of these networks show evoked persistent activity in an all-or-none manner. Blocking inhibition with bicuculline methaiodide (BMI) leads to an increase in the duration of persistent activity. These persistent phases were abolished by blockade of asynchronous neurotransmitter release by ethylene glycol tetraacetic acid (EGTA-AM).     

Systemic LPS administration induces brain inflammation but not dopaminergic neuronal death in the substantia nigra

It has been suggested that brain inflammation is important in aggravation of brain damage and/or that inflammation causes neurodegenerative diseases including Parkinson's disease (PD). Recently, systemic inflammation has also emerged as a risk factor for PD. In the present study, we evaluated how systemic inflammation induced by intravenous (iv) lipopolysaccharides (LPS) injection affected brain inflammation and neuronal damage in the rat. Interestingly, almost all brain inflammatory responses, including morphological activation of microglia, neutrophil infiltration, and mRNA/protein expression of inflammatory mediators, appeared within 4-8 h, and subsided within 1-3 days, in the substantia nigra (SN), where dopaminergic neurons are located. More importantly, however, dopaminergic neuronal loss was not detectable for up to 8 d after iv LPS injection. Together, these results indicate that acute induction of systemic inflammation causes brain inflammation, but this is not sufficiently toxic to induce neuronal injury.     

Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells—especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.     

Identification of the neuronal effects of ethanol on <Emphasis Type="Italic">C. elegans</Emphasis> by in vivo fluorescence imaging on a microfluidic chip

Caenorhabditis elegans (C. elegans) is a well-established model organism for investigating the correlations between behavioral and neuronal activities. Here, we demonstrated a microfluidic-based method that allowed stimulation-based neuronal analysis of immobilized C. elegans for identifying the neuronal effects of ethanol on the chemosensory responses of the right ASE (ASER) neuron. A one-piece microvalve was developed for the immobilization of C. elegans. Stimulations were realized by interface shifting of laminar flows. Well-fed transgenic worms expressing the calcium indicator G-CaMP in ASER neurons were used for in vivo fluorescence imaging. To evaluate the developed method, we first studied the effects of ethanol on the ASER neurons in response to a single NaCl stimulus. Results indicated that ethanol acutely suppressed the ON responses of ASER neurons to NaCl rather than the OFF response. Further studies of the adaptation of ASER neurons in response to NaCl and in the presence of ethanol suggested that ethanol interfered with the adaptation of neurons. The developed method exhibited the advantages of ease of operation and high throughput. We expect this new method to open up a new avenue for investigating the correlations between the behavioral and neuronal activities of C. elegans.     

The role of bioactive compounds on the promotion of neurite outgrowth

Neurite loss is one of the cardinal features of neuronal injury. Apart from neuroprotection, reorganization of the lost neuronal network in the injured brain is necessary for the restoration of normal physiological functions. Neuritogenic activity of endogenous molecules in the brain such as nerve growth factor is well documented and supported by scientific studies which show innumerable compounds having neurite outgrowth activity from natural sources. Since the damaged brain lacks the reconstructive capacity, more efforts in research are focused on the identification of compounds that promote the reformation of neuronal networks. An abundancy of natural resources along with the corresponding activity profiles have shown promising results in the field of neuroscience. Recently, importance has also been placed on understanding neurite formation by natural products in relation to neuronal injury. Arrays of natural herbal products having plentiful active constituents have been found to enhance neurite outgrowth. They act synergistically with neurotrophic factors to promote neuritogenesis in the diseased brain. Therefore use of natural products for neuroregeneration provides new insights in drug development for treating neuronal injury. In this study, various compounds from natural sources with potential neurite outgrowth activity are reviewed in experimental models.     

Genetically-encoded voltage indicators

Voltage imaging with genetically-encoded sensors has allowed for the direct visualization of electrical signaling at high spatial resolutions. Over the history of voltage indicator development, various design strategies have been employed to harness the power of the fl uctuating transmembrane electric fi eld.     

基于碳纳米材料的神经电极技术

As a powerful tool for monitoring and modulating neural activities, implantable neural electrodes constitute the basis for a wide range of applications, including fundamental studies of brain circuits and functions, treatment of various neurological diseases, and realization of brain-machine interfaces. However, conventional neural electrodes have the issue of mechanical mismatch with soft neural tissues, which can result in tissue inflammation and gliosis, thus causing degradation of function over chronic implantation. Furthermore, implantable neural electrodes, especially depth electrodes, can only carry out limited data sampling within predefined anatomical regions, making it challenging to perform large-area brain mapping. With excellent electrical, mechanical, and chemical properties, carbon-based nanomaterials, including graphene and carbon nanotubes (CNTs), have been used as materials of implantable neural electrodes in recent years. Electrodes made from graphene and CNT fibers exhibit low electrochemical impedance, benefiting from the porous microstructure of the fibers. This enables a much smaller size of neural electrode. Together with the low Young's modulus of the fibers, this small size results in very soft electrodes. Soft neural electrodes made from graphene and CNT fibers show a much-reduced inflammatory response and enable stable chronic in vivo action potential recording for 4-5 months. Combining different modalities of neural interfacing, including electrophysiological measurement, optical imaging/stimulation, and magnetic resonance imaging (MRI), could leverage the spatial and temporal resolution advantages of different techniques, thus providing new insights into how neural circuits process information. Transparent neural electrode arrays made from graphene or CNTs enable simultaneous calcium imaging through the transparent electrodes, from which concurrent electrical recording is taken, thus providing complementary cellular information in addition to high-temporal-resolution electrical recording. Transparent neural electrodes from carbon-based nanomaterials can record well-defined neuronal response signals with negligible light-induced artifacts from cortical surfaces under optogenetic stimulation. Graphene and CNT-based materials were used to fabricate MRI-compatible neural electrodes with negligible artifacts under high field MRI. Simultaneous deep brain stimulation (DBS) and functional magnetic resonance imaging (fMRI) with graphene fiber electrodes in the subthalamic nucleus (STN) in Parkinsonian rats revealed robust blood oxygenation level dependent responses along the basal ganglia-thalamocortical network in a frequency-dependent manner, with responses from some regions not previously detectable. This review introduces the recent development and application of neural electrode technologies based on graphene and CNTs. We also discuss biological safety issues and challenges faced by neural electrodes made from carbon nanomaterials. The use of carbon-based nanomaterials for the fabrication of various soft and multi-modality compatible neural electrodes will provide a powerful platform for both fundamental and translational neuroscience research.     

Modelling of calcium dynamics in brain energy metabolism and Alzheimer's disease

Functional imaging techniques play a major role in the study of brain activation by monitoring the changes in blood flow and energy metabolism. In order to interpret functional neuroimaging data better, the existing mathematical models describing the links that may exist between electrical activity, energy metabolism and hemodynamics in literature are thoroughly analyzed for their advantages and disadvantages in terms of their prediction of available experimental data. Then, these models are combined within a single model that includes membrane ionic currents, glycolysis, mitochondrial activity, exchanges through the blood-brain barrier, as well as brain hemodynamics. Particular attention is paid to the transport and storage of calcium ions in neurons since calcium is not only an important molecule for signalling in neurons, but it is also essential for memory storage. Multiple efforts have underlined the importance of calcium dependent cellular processes in the biochemical characterization of Alzheimer's disease (AD), suggesting that abnormalities in calcium homeostasis might be involved in the pathophysiology of the disease. The ultimate goal of this study is to investigate the hypotheses about the physiological or biochemical changes in health and disease and to correlate them to measurable physiological parameters obtained from functional neuroimaging data as in the time course of blood oxygenation level dependent (BOLD) signal. When calcium dynamics are included in the model, both BOLD signal and metabolite concentration profiles are shown to exhibit temporal behaviour consistent with the experimental data found in literature. In the case of Alzheimer's disease, the effect of halved cerebral blood flow increase results in a negative BOLD signal implying suppressed neural activity.     

Design and synthesis of type-III mimetics of omega-conotoxin GVIA

Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. Our approach involves interactive design of conformationally defined molecular scaffolds that project certain functional groups in a way that mimics the projection of important binding residues as determined in the parent structure. These design principles are discussed and applied to the structurally defined polypeptide, -conotoxin GVIA, which blocks voltage-gated, neuronal N-type calcium channels. These ion channels represent therapeutic targets for the development of new analgesics that can treat chronic pain. It is shown how a discontinuous, 3-residue pharmacophore of GVIA can be mimicked by different molecular scaffolds. It is illustrated how such 1^st generation leads must necessarily be weak and that optimisability must therefore be built-in during the design process.     

NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling

To address existing limitations in live neuron imaging, we have developed NeuO , a novel cell‐permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.     

High pressure effects in anaesthesia and narcosis

There is growing interest in determining the effects of high pressure on biological functions. Studies of brain processes under hyperbaric conditions can give a unique insight into phenomena such as nitrogen narcosis, inert gas anaesthesia, and pressure reversal of the effects of anaesthetic and narcotic agents. Such research may shed light on the action of anaesthetics, which remains poorly understood, and on the nature of consciousness itself. Various studies have established the behavioural response of organisms to hyperbaric conditions, in the presence or absence of anaesthetic agents. At the molecular level, X-ray crystallography has been used to investigate the incorporation of species like Xe in hydrophobic pockets within model ion channels that may account for pressure effects on neuronal transmission. New magnetic resonance imaging techniques are providing tomographic three-dimensional images that detail brain structure and function, and that can be correlated with behavioural studies and psychological test results. Such whole organ techniques are linked to the molecular scale via voltage-sensitive dye (VSD) imaging studies on brain slices that provide time-resolved images of the dynamic formation and interconnection of inter-neuronal complexes. The VSD experiments are readily adapted to in situ studies under high pressure conditions. In this tutorial review we review the current state of knowledge of hyperbaric effects on brain processes: anaesthesia and narcosis, recent studies at the molecular level via protein crystallography at high pressure in a Xe atmosphere, and we also present some preliminary results of VSD imaging of brain slices under hyperbaric conditions.     

基于微电极阵列的睡眠剥夺大鼠海马区神经电活动检测

Sleep deprivation (SD) is the partial or complete loss of sleep and has long been used as a tool in sleep research to interfere with normal sleep cycles in rodents and humans. The progressively-accumulating sleep pressure induced by sleep deprivation can lead to a variety of physiological changes and even death. Compared to traditional detection methods, in vivo detection of neuronal activity using micro-electromechanical system (MEMS) technology following sleep deprivation can help fully elucidate the effects of sleep deprivation at the cellular level. Herein, a computer-controlled rotary roller was used to completely deprive rats of sleep for 14 days and 16-channel microelectrode arrays (MEAs) were fabricated and implanted into the rat hippocampus to measure neural spikes and local field potentials (LFPs) in real-time. The hippocampus is involved in learning and memory and has been the focus of intensive research aimed at understanding the function of sleep. This study was performed to measure the changes in neuronal activity in the rat hippocampus induced by sleep deprivation as well as their overall impact on the brain. After sleep deprivation, both the pyramidal- and inter-neurons showed a higher amplitude and more intense firing patterns. The fast-firing pattern of the neurons after sleep deprivation indicated elevated excitability in the prolonged awake state. In addition, the LFP of the sleep deprived rats fluctuated more frequently. The power of the LFPs in the low-frequency band (0–50 Hz) was calculated, showing increased power of the delta, theta, alpha, and beta bands after sleep deprivation, especially for the delta band (0.1–4 Hz). Generally, LFPs are generated by all types of neural activity in the neural circuit, and the changes in the low frequency band power suggested decreased arousal and increased sleep pressure induced by sleep deprivation, which could further impair brain function. This study was mainly aimed at measuring electrophysiological changes induced by sleep deprivation in the rat brain. Typically, neuronal activity changes were accompanied by the alternation of specific neurotransmitters in the brain. In the future, it will be essential to focus on measuring the concurrent change of electrophysiological and neurochemical signals to better examine the impact of sleep deprivation on brain function.     

Production, quality control and pharmacokinetic studies of 177Lu–zoledronate for bone pain palliation therapy

A new dipeptide derivative, ethyl 2-(3-(4-hydroxyphenyl)-2-(2-(4-phenyl-5-((pyridin-4-ylamino)methyl)-4H-1,2,4-triazol-3-ylthio)acetamido)propanamido)-3-(1H-indol-3-yl)propanoate (EHPTIP) was successfully synthesized and radiolabeled with ¹²⁵I by the direct electrophilic substitution method. The non radiolabeled compound (EHPTIP) was tested as an antimicrobial agent and the radiolabeled derivative was tested as a new imaging agent. The study results showed a good antimicrobial activity of EHPTIP and a good in vitro and in vivo stability of ¹²⁵I-EHPTIP. The biodistribution of the radiolabeled compound showed a high brain uptake of 7.60 ± 0.01 injected activity/g tissue organ at 30 min post-injection and retention in brain remained high up to 1 h, whereas the clearance from the normal mice appeared to proceed via the renal system. Such brain uptake is better than that of currently used radiopharmaceuticals for brain imaging (^99mTc-ECD and ^99mTc-HMPAO). As a conclusion, EHPTIP is a newly synthesized dipeptide with a good antimicrobial activity and the radioiodinated EHPTIP which is labeled with ¹²³I could be used as a novel agent for brain SPECT.     

Visualization of Intra‐neuronal Motor Protein Transport through Upconversion Microscopy

Cargo transport along axons, a physiological process mediated by motor proteins, is essential for neuronal function and survival. A current limitation in the study of axonal transport is the lack of a robust imaging technique with a high spatiotemporal resolution to visualize and quantify the movement of motor proteins in real‐time and in different depth planes. Herein, we present a dynamic imaging technique that fully exploits the characteristics of upconversion nanoparticles. This technique can be used as a microscopic probe for the quantitative in situ tracking of retrograde transport neurons with single‐particle resolution in multilayered cultures. This study may provide a powerful tool to reveal dynamic neuronal activity and intra‐axonal transport function as well as any associated neurodegenerative diseases resulting from mutation or impairment in the axonal transport machinery.     

Green Tea Suppresses Brain Aging

Epidemiological studies have demonstrated that the intake of green tea is effective in reducing the risk of dementia. The most important component of green tea is epigallocatechin gallate (EGCG). Both EGCG and epigallocatechin (EGC) have been suggested to cross the blood–brain barrier to reach the brain parenchyma, but EGCG has been found to be more effective than EGC in promoting neuronal differentiation. It has also been suggested that the products of EGCG decomposition by the intestinal microbiota promote the differentiation of nerve cells and that both EGCG and its degradation products act on nerve cells with a time lag. On the other hand, the free amino acids theanine and arginine contained in green tea have stress-reducing effects. While long-term stress accelerates the aging of the brain, theanine and arginine suppress the aging of the brain due to their anti-stress effect. Since this effect is counteracted by EGCG and caffeine, the ratios between these green tea components are important for the anti-stress action. In this review, we describe how green tea suppresses brain aging, through the activation of nerve cells by both EGCG and its degradation products, and the reductions in stress achieved by theanine and arginine.     

Raman spectroscopic imaging for in vivo detection of cerebral brain metastases

We report for the first time a proof-of-concept experiment employing Raman spectroscopy to detect intracerebral tumors in vivo by brain surface mapping. Raman spectroscopy is a non-destructive biophotonic method which probes molecular vibrations. It provides a specific fingerprint of the biochemical composition and structure of tissue without using any labels. Here, the Raman system was coupled to a fiber-optic probe. Metastatic brain tumors were induced by injection of murine melanoma cells into the carotid artery of mice, which led to subcortical and cortical tumor growth within 14 days. Before data acquisition, the cortex was exposed by creating a bony window covered by a calcium fluoride window. Spectral contributions were assigned to proteins, lipids, blood, water, bone, and melanin. Based on the spectral information, Raman images enabled the localization of cortical and subcortical tumor cell aggregates with accuracy of roughly 250 μm. This study demonstrates the prospects of Raman spectroscopy as an intravital tool to detect cerebral pathologies and opens the field for biophotonic imaging of the living brain. Future investigations aim to reduce the exposure time from minutes to seconds and improve the lateral resolution.