Membrane Proteome-Wide Screening of Autoantibodies in CIDP Using Human Cell Microarray Technology.

BACKGROUND AND OBJECTIVES: Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. RESULTS: Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. DISCUSSION: Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.

Differential regulations of neural activity and survival in primary cortical neurons by PFOA or PFHpA.

Perfluorinated compounds (PFCs), organofluoride compounds comprising carbon-fluorine and carbon-carbon bonds, are used as water and oil repellents in textiles and pharmaceutical tablets; however, they are associated with potential neurotoxic effects. Moreover, the impact of PFCs on neuronal survival, activity, and regulation within the brain remains unclear. Additionally, the mechanisms through which PFCs induce neuronal toxicity are not well-understood because of the paucity of data. This study elucidates that perfluorooctanoic acid (PFOA) and perfluoroheptanoic acid (PFHpA) exert differential effects on the survival and activity of primary cortical neurons. Although PFOA triggers apoptosis in cortical neurons, PFHpA does not exhibit this effect. Instead, PFHpA modifies dendritic spine morphogenesis and synapse formation in primary cortical neuronal cultures, additionally enhancing neural activity and synaptic transmission. This research uncovers a novel mechanism through which PFCs (PFHpA and PFOA) cause distinct alterations in dendritic spine morphogenesis and synaptic activity, shedding light on the molecular basis for the atypical behaviors noted following PFC exposure. Understanding the distinct effects of PFHpA and PFOA could guide regulatory policies on PFC usage and inform clinical approaches to mitigate their neurotoxic effects, especially in vulnerable population.

A tool kit of highly selective and sensitive genetically encoded neuropeptide sensors.

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution. However, this has been hindered by the lack of direct, sensitive, and noninvasive tools. We developed a series of GRAB (G protein-coupled receptor activation‒based) sensors for detecting somatostatin (SST), corticotropin-releasing factor (CRF), cholecystokinin (CCK), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors, which enable detection of specific neuropeptide binding at nanomolar concentrations, establish a robust tool kit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

Neural System Impairment and Involved Microglia-Neuron Regulation of Broflanilide in Zebrafish Larvae.

Broflanilide is widely used to control pests and has attracted attention due to its adverse effects on aquatic organisms. Our previous study showed that broflanilide has a negative impact on the central nervous system (CNS) at lethal dosages; however, its neural effects under practical situations and the underlying mechanisms remain unknown. To elucidate how broflanilide affects the CNS, we exposed zebrafish larvae to broflanilide at 16.9 and 88.0 µg/L (the environmentally relevant concentrations) for 120 h. Zebrafish locomotion was significantly disturbed at 88.0 µg/L, with a decreased moving distance and velocity accompanied by an inhibited neurotransmitter level. In vivo neuroimaging analysis indicated that the nerves of zebrafish larvae, including the axons, myelin sheaths, and neurons, were impaired. The number of neurons was significantly reduced after exposure, with an impaired morphological structure. These changes were accompanied by the abnormal transcription of genes involved in early CNS development. In addition, an increased total number of microglia and an elevated proportion of amoeboid microglia were observed after 88.0 µg/L broflanilide exposure, pointing out to an upstream role of microglia activation in mediating broflanilide neurotoxicity. Meanwhile, increased inflammatory cytokine levels and brain neutrophil numbers were observed, implicating significant inflammatory response and immune toxicity. Our findings indicate that broflanilide interferes with microglia-neuron regulation and induces neurodevelopmental disorders.

Loss of Ephaptic Contacts in the Murine Thalamus during Osmotic Demyelination Syndrome.

BACKGROUND AND AIM: A murine model mimicking osmotic demyelination syndrome (ODS) revealed with histology in the relay posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei adjoined nerve cell bodies in chronic hyponatremia, amongst the damaged 12 h and 48 h after reinstatement of osmolality. This report aims to verify and complement with ultrastructure other neurophysiology, immunohistochemistry, and molecular biochemistry data to assess the connexin-36 protein, as part of those hinted close contacts.This ODS investigation included four groups of mice: Sham (NN; n = 13), hyponatremic (HN; n = 11), those sacrificed 12 h after a fast restoration of normal natremia (ODS12h; n = 6) and mice sacrificed 48 h afterward, or ODS48 h (n = 9). Out of these, thalamic zones samples included NN (n = 2), HN (n = 2), ODS12h (n = 3) and ODS48h (n = 3). RESULTS: Ultrastructure illustrated junctions between nerve cell bodies that were immunolabeled with connexin36 (Cx36) with light microscopy and Western blots. These cell's junctions were reminiscent of low resistance junctions characterized in other regions of the CNS with electrophysiology. Contiguous neurons showed neurolemma contacts in intact and damaged tissues according to their location in the ODS zones, at 12 h and 48 h post correction along with other demyelinating alterations. Neurons and ephaptic contact measurements indicated the highest alterations, including nerve cell necrosis in the ODS epicenter and damages decreased toward the outskirts of the demyelinated zone. CONCLUSION: Ephapses contained C × 36between intact or ODS injured neurons in the thalamus appeared to be resilient beyond the core degraded tissue injuries. These could maintain intercellular ionic and metabolite exchanges between these lesser injured regions and, thus, would partake to some brain plasticity repairs.

Optoelectronic graded neurons for bioinspired in-sensor motion perception.

Motion processing has proven to be a computational challenge and demands considerable computational resources. Contrast this with the fact that flying insects can agilely perceive real-world motion with their tiny vision system. Here we show that phototransistor arrays can directly perceive different types of motion at sensory terminals, emulating the non-spiking graded neurons of insect vision systems. The charge dynamics of the shallow trapping centres in MoS2 phototransistors mimic the characteristics of graded neurons, showing an information transmission rate of 1,200 bit s-1 and effectively encoding temporal light information. We used a 20 × 20 photosensor array to detect trajectories in the visual field, allowing the efficient perception of the direction and vision saliency of moving objects and achieving 99.2% recognition accuracy with a four-layer neural network. By modulating the charge dynamics of the shallow trapping centres of MoS2, the sensor array can recognize motion with a temporal resolution ranging from 101 to 106 ms.

Morphology and immunohistochemical characteristics of the otic ganglion in the chinchilla (Chinchilla laniger Molina).

INTRODUCTION: The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding. MATERIAL AND METHODS: Morphological investigations of the otic ganglion of the chinchilla were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with the double-labelling immunofluorescence method. RESULTS: Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40-50 µm) accounted for about 80% of the neurons in the cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT+ and NOS+ neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS+ only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalins, substance P, somatostatin, and galanin was expressed in single nerve cell bodies and nerve fibers except numerous substance P+ intraganglionic nerve fibers. Some of them were stained also for CGRP. Single neurons stained for tyroxine hydroxylase. CONCLUSIONS: Our results, compared with findings in other rodent species suggest the existence of interspecies differences in the morphology, cellular structure, and immunohistochemical properties of the head autonomic ganglia in mammals.

Real-Time Monitoring of Neurotransmitters in the Brain of Living Animals.

Neurotransmitters, as important chemical small molecules, perform the function of neural signal transmission from cell to cell. Excess concentrations of neurotransmitters are often closely associated with brain diseases, such as Alzheimer's disease, depression, schizophrenia, and Parkinson's disease. On the other hand, the release of neurotransmitters under the induced stimulation indicates the occurrence of reward-related behaviors, including food and drug addiction. Therefore, to understand the physiological and pathological functions of neurotransmitters, especially in complex environments of the living brain, it is urgent to develop effective tools to monitor their dynamics with high sensitivity and specificity. Over the past 30 years, significant advances in electrochemical sensors and optical probes have brought new possibilities for studying neurons and neural circuits by monitoring the changes in neurotransmitters. This Review focuses on the progress in the construction of sensors for in vivo analysis of neurotransmitters in the brain and summarizes current attempts to address key issues in the development of sensors with high selectivity, sensitivity, and stability. Combined with the latest advances in technologies and methods, several strategies for sensor construction are provided for recording chemical signal changes in the complex environment of the brain.

Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin. METHODS: Human choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein-gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used. RESULTS: In choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity. CONCLUSION: UCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Upregulation of Connexins in the Rat Hippocampal and Cortical Neurons Following Blockade of NMDA Receptors During Postnatal Development.

BACKGROUND: Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3. METHODS: Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group. RESULTS: Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors. CONCLUSION: These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells.

A Push-Pull Mechanism Between PRRT2 and ß4-subunit Differentially Regulates Membrane Exposure and Biophysical Properties of NaV1.2 Sodium Channels.

Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.

Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles.

Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain-Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones' previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry-based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.

Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes.

Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm3, whereas this density has a very low viral titer in the prototypic Fermon strain. IMPORTANCE Despite the strong causal link between enterovirus D68 (EV-D68) and acute flaccid myelitis (AFM), it remains unclear how EV-D68 gains entry into the central nervous system and what receptors enable it to infect motor neurons. We show that EV-D68 particles can adhere to exosomes, placing EV-D68 among a handful of other picornaviruses which are known to interact with extracellular vesicles. Uptake and infection of permissive cells by virally contaminated exosomes would have major implications in the search for the EV-D68 receptor, as well as providing a possible route for viral entry into motor neurons. This work identifies a novel cellular entry route for EV-D68 and may facilitate the identification of genetic risk factors for development of AFM.

Scalable mapping of myelin and neuron density in the human brain with micrometer resolution.

Optical coherence tomography (OCT) is an emerging 3D imaging technique that allows quantification of intrinsic optical properties such as scattering coefficient and back-scattering coefficient, and has proved useful in distinguishing delicate microstructures in the human brain. The origins of scattering in brain tissues are contributed by the myelin content, neuron size and density primarily; however, no quantitative relationships between them have been reported, which hampers the use of OCT in fundamental studies of architectonic areas in the human brain and the pathological evaluations of diseases. Here, we built a generalized linear model based on Mie scattering theory that quantitatively links tissue scattering to myelin content and neuron density in the human brain. We report a strong linear relationship between scattering coefficient and the myelin content that is retained across different regions of the brain. Neuronal cell body turns out to be a secondary contribution to the overall scattering. The optical property of OCT provides a label-free solution for quantifying volumetric myelin content and neuron cells in the human brain.

Quantifying phosphorylation dynamics in primary neuronal cultures using LC-MS/MS.

Cellular processes require tight and coordinated control of protein abundance, localization, and activity. One of the core mechanisms to achieve specific regulation of proteins is protein phosphorylation. Here we present a workflow to monitor protein abundance and phosphorylation in primary cultured neurons using liquid chromatography-coupled mass spectrometry. Our protocol provides a detailed guide on all steps for detection and label-free-quantification of phosphorylated and unmodified proteins of primary cortical neurons, including primary cell culture, phosphoproteomic sample preparation and data-processing, and evaluation. For complete details on the use and execution of this protocol, please refer to Desch et al. (2021).

Co-existing Neuropeptide FF and Gonadotropin-Releasing Hormone 3 Coordinately Modulate Male Sexual Behavior.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertoires were delayed, not disrupted, in gnrh3 and npff single knockout males, while the double knockout appeared to alleviate the significant defects that were observed in single knockouts. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which co-existing peptides of the TN-GnRH neurons, NPFF, and GnRH3 coordinately modulate certain neuronal circuit for the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to sensory information from the external environments.

Optical tools to study the subcellular organization of GPCR neuromodulation.

Modulation of neuronal circuit activity is key to information processing in the brain. G protein-coupled receptors (GPCRs), the targets of most neuromodulatory ligands, show extremely diverse expression patterns in neurons and receptors can be localized in various sub-neuronal membrane compartments. Upon activation, GPCRs promote signaling cascades that alter the level of second messengers, drive phosphorylation changes, modulate ion channel function, and influence gene expression, all of which critically impact neuron physiology. Because of its high degree of complexity, this form of interneuronal communication has remained challenging to integrate into our conceptual understanding of brain function. Recent technological advances in fluorescence microscopy and the development of optical biosensors now allow investigating neuromodulation with unprecedented resolution on the level of individual cells. In this review, we will highlight recent imaging techniques that enable determining the precise localization of GPCRs in neurons, with specific focus on the subcellular and nanoscale level. Downstream of receptors, we describe novel conformation-specific biosensors that allow for real-time monitoring of GPCR activation and of distinct signal transduction events in neurons. Applying these new tools has the potential to provide critical insights into the function and organization of GPCRs in neuronal cells and may help decipher the molecular and cellular mechanisms that underlie neuromodulation.

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes.

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

Metabolic Labeling of Primary Neurons Using Carbohydrate Click Chemistry.

Glycans play an important role in many neuronal processes, such as neurotransmitter release and reuptake, cell-cell communication and adhesion, modulation of ion channel activity, and immune function. Carbohydrate click chemistry is a powerful technique for studying glycan function and dynamics in vitro, in vivo, and ex vivo. Here, we use commercially available synthetic tetraacetylated azido sugars, copper and copper-free click chemistry to metabolically label and analyze primary rat cortical neurons. In addition, we use high resolution confocal and STED microscopy to image and analyze different forms of glycosylation in ultrahigh resolution. We observe different patterns of GlcNAz, GalNAz, and ManNAz distribution at different stages of neuronal development. We also observe highly sialylated structures on the neuronal plasma membrane, which warrant further investigation.

Rapid Fluorescence Lifetime Imaging Reveals That TRPV4 Channels Promote Dysregulation of Neuronal Na+ in Ischemia.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.