Mesoscale standing wave imaging.

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high-magnification, high-numerical aperture objective lenses, and while this results in high-resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low-magnification and high-numerical aperture.

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines.

Rationale: Major depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies.

Objectives: In the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour.

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems.

Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS).

Use of sedative-hypnotic medications and risk of dementia: A systematic review and meta-analysis.

Aims: Growing evidence suggests an association between the use of sedative-hypnotic medications and risk of dementia. The aim of this study is to examine this association using a meta-analysis approach.

Methods: MEDLINE (PubMed) and Scopus were systematically searched for studies published in English only.

Increased Levels of IL-16 in the Central Nervous System during Neuroinflammation Are Associated with Infiltrating Immune Cells and Resident Glial Cells.

Interleukin (IL)-16, a CD4 immune cell specific chemoattractant cytokine, has been shown to be involved in the development of multiple sclerosis, an inflammatory demyelinating disease of the central nervous system (CNS). While immune cells such as T cells and macrophages are reported to be the producers of IL-16, the cellular source of IL-16 in the CNS is less clear. This study investigates the correlation of IL-16 expression levels in the CNS with the severity of neuroinflammation and determines the phenotype of cells which produce IL-16 in the CNS of experimental autoimmune encephalomyelitis (EAE) mice.

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton.

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart.

The development of proteinase-activated receptor-2 modulators and the challenges involved.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period.

Mitogen-activated protein kinase phosphatase-2 deletion modifies ventral tegmental area function and connectivity and alters reward processing.

Mitogen-activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation.

Interleukin-16 inhibits sodium channel function and GluA1 phosphorylation via CD4- and CD9-independent mechanisms to reduce hippocampal neuronal excitability and synaptic activity.

Interleukin 16 (IL-16) is a cytokine that is primarily associated with CD4 T cell function, but also exists as a multi-domain PDZ protein expressed within cerebellar and hippocampal neurons. We have previously shown that lymphocyte-derived IL-16 is neuroprotective against excitotoxicity, but evidence of how it affects neuronal function is limited. Here, we have investigated whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippocampal cultures.

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy.

We present here a fast optical sectioning method for mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm × 3 mm × 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.

The therapeutic effect of anti-CD52 treatment in murine experimental autoimmune encephalomyelitis is associated with altered IL-33 and ST2 expression levels.

Experimental autoimmune encephalomyelitis (EAE) mice were administered with murine anti-CD52 antibody to investigate its therapeutic effect and whether the treatment modulates IL-33 and ST2 expression. EAE severity and central nervous system (CNS) inflammation were reduced following the treatment, which was accompanied by peripheral T and B lymphocyte depletion and reduced production of various cytokines including IL-33, while sST2 was increased. In spinal cords of EAE mice, while the number of IL-33 cells remained unchanged, the extracellular level of IL-33 protein was significantly reduced in anti-CD52 antibody treated mice compared with controls.

Toll-like receptor 3 activation impairs excitability and synaptic activity via TRIF signalling in immature rat and human neurons.

Toll like receptor 3 (TLR3) belongs to a family of pattern recognition receptors that recognise molecules found on pathogens referred to as pathogen associated molecular patterns (PAMPs). Its involvement in innate immunity is well known but despite its presence in the central nervous system (CNS), our knowledge of its function is limited. Here, we have investigated whether TLR3 activation modulates synaptic activity in primary hippocampal cultures and induced pluripotent stem cell (iPSC)-derived neurons.

A Microfluidic Platform for the Characterisation of CNS Active Compounds.

New in vitro technologies that assess neuronal excitability and the derived synaptic activity within a controlled microenvironment would be beneficial for the characterisation of compounds proposed to affect central nervous system (CNS) function. Here, a microfluidic system with computer controlled compound perfusion is presented that offers a novel methodology for the pharmacological profiling of CNS acting compounds based on calcium imaging readouts. Using this system, multiple applications of the excitatory amino acid glutamate (10 nM-1 mM) elicited reproducible and reversible transient increases in intracellular calcium, allowing the generation of a concentration response curve.

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility.

GABA receptors suppress burst-firing in reticular thalamic neurons.

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN.

Proteinase-activated receptor 2 is involved in the behavioural changes associated with sickness behaviour.

Proteinase-activated receptor-2 (PAR2) is widely expressed in the CNS but whether it plays a key role in inflammation-related behavioural changes remains unknown. Hence, in the present study we have examined whether PAR2 contributes to behaviour associated with systemic inflammation using PAR2 transgenic mice. The onset of sickness behaviour was delayed and the recovery accelerated in PAR2(-/-) mice in the LPS-induced model of sickness behaviour.

Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation.

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca(2+) events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca(2+) indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Protease-Activated Receptor 2: Are Common Functions in Glial and Immune Cells Linked to Inflammation-Related CNS Disorders?

Protease-activated receptors (PARs) are a novel family of G-protein coupled receptors (GPCRs) whose activation requires the cleavage of the N-terminus by a serine protease. However, recent evidence reveals that alternative routes of activation also occur, that PARs signal via multiple pathways and that pathway activation is activator- dependent. Given our increased understanding of PAR function both under physiological and pathophysiological conditions, one aspect that has remained constant is the link between PAR2 and inflammation.

Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease.

Chemically induced synaptic activity between mixed primary hippocampal co-cultures in a microfluidic system.

Primary neuronal cultures are an invaluable in vitro tool for examining the fundamental physiological changes that occur in diseases of the central nervous system. In this work, we have used a microfluidic device to grow twin cultures of primary hippocampal neuronal/glia cells which are synaptically connected but environmentally isolated. Immunocytochemical staining, for β-III-Tubulin and synaptophysin, indicated that the two neuronal populations were physically connected and that synapses were present.

Lymphocyte-mediated neuroprotection in in vitro models of excitotoxicity involves astrocytic activation and the inhibition of MAP kinase signalling pathways.

It is well established that immunosurveillance is active in the CNS and plays a key role in several CNS disorders but the exact role of immune cells remains elusive. Thus, in the present study we investigated whether lymphocytes are protective/detrimental in in vitro models of excitotoxicty. Kainate (KA)-induced neuronal death was significantly reduced following exposure to mixed lymphocytes or purified T lymphocytes containing either activated or non-activated T-lymphocytes.

In vivo real-time multiphoton imaging of T lymphocytes in the mouse brain after experimental stroke.

Background And Purpose: To gain a better understanding of T cell behavior after stroke, we have developed real-time in vivo brain imaging of T cells by multiphoton microscopy after middle cerebral artery occlusion.

Methods: Adult male hCD2-GFP transgenic mice that exhibit green fluorescent protein-labeled T cells underwent permanent left distal middle cerebral artery occlusion by electrocoagulation (n=6) or sham surgery (n=6) and then multiphoton laser imaging 72 hours later.

Results: Extravasated T cell number significantly increased after middle cerebral artery occlusion versus sham.