Single-cell Calcium Imaging Research Articles

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Might synthetic cannabinoids influence neural differentiation?

Phytocannabinoids are psychotropic substances found in cannabis that bind to the endocannabinoid receptors regulating a variety of physiological processes in human body, including synaptic activity in the central nervous system and metabolic effects in the peripheric nervous system among many others [1,2]. Synthetic cannabinoids emerged as popular alternative to cannabis. Most of these substances are synthetic analogues of Δ9-THC, the psychotropic compound of cannabis, binding with higher affinity to the endocannabinoid receptor CB1 and eliciting a stronger and long-lasting effect on brain cells. Molecular structure of synthetic cannabinoids is always changing escaping the control by authorities and increasing the hazard for general population. The popularity of cannabis and its derivatives may lead, and often does, to child’s exposure to cannabinoids both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with higher risk of newborn morbidity [2,3], altered rate of mental development and significant changes in nervous system functioning [4,5]. However, direct evidence that these effects are mediated through the binding of cannabinoids to endocannabinoid receptors is still lacking. Thus, it is paramount to better understand the psychoactive effects of natural and synthetic cannabinoids on the developing human brain. We conveyed a pilot study in which human induced pluripotent stem cells (hiPSCs) were induced into neural differentiation and treated with a non-psychotropic component of cannabis, cannabidiol, known to bind the CB2 receptor, and two synthetic Δ9-THC analogues, THJ-018 and EG-018. Neuronal differentiation and functional maturation were assessed by immunofluorescence, qRT-PCR and single cell calcium imaging. Our results indicate that all three substances have profound impact on the differentiation, maturation and functioning of developing CNS neurons, providing a new evidence for the importance of thorough research of the impact of pre-natal exposure to natural and synthetic cannabinoids.

Expression and Role of Thyrotropin Receptors in Proopiomelanocortin-Producing Pituitary Cells.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer’s disease

Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 μM, 3 μM and 10 μM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

Cell Calcium Imaging as a Reliable Method to Study Neuron-Glial Circuits.

Complex dynamic cellular networks have been studied in physiological and pathological processes under the light of single-cell calcium imaging (SCCI), a method that correlates functional data based on calcium shifts operated by different intracellular and extracellular mechanisms integrated with their cell phenotypes. From the classic synaptic structure to tripartite astrocytic model or the recent quadripartite microglia added ensemble, as well as other physiological tissues, it is possible to follow how cells signal spatiotemporally to cellular patterns. This methodology has been used broadly due to the universal properties of calcium as a second messenger. In general, at least two types of receptor operate through calcium permeation: a fast-acting ionotropic receptor channel and a slow-activating metabotropic receptor, added to exchangers/transporters/pumps and intracellular Ca2+ release activated by messengers. These prototypes have gained an enormous amount of information in dynamic signaling circuits. SCCI has also been used as a method to associate phenotypic markers during development and stage transitions in progenitors, stem, vascular cells, neuro- and glioblasts, neurons, astrocytes, oligodendrocytes, and microglia that operate through ion channels, transporters, and receptors. Also, cancer cells or inducible cell lines from human organoids characterized by transition stages are currently being used to model diseases or reconfigure healthy cells in terms of the expression of calcium-binding/permeable molecules and shed light on therapy.

Human Pluripotent Stem Cell-Derived Neurons Are Functionally Mature In Vitro and Integrate into the Mouse Striatum Following Transplantation

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely “high”, “intermediate” and “low” firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology.

High-throughput screening of human induced pluripotent stem cell-derived brain organoids

BackgroundThe need for scalable high-throughput screening (HTS) approaches for 3D human stem cell platforms remains a central challenge for disease modeling and drug discovery. We have developed a workflow to screen cortical organoids across platforms. New methodWe used serum-free embryoid bodies (SFEBs) derived from human induced pluripotent stem cells (hiPSCs) and employed high-content imaging (HCI) to assess neurite outgrowth and cellular composition within SFEBs. We multiplexed this screening assay with both multi-electrode arrays (MEAs) and single-cell calcium imaging. ResultsHCI was used to assess the number of excitatory neurons (VGlut+) in experimental replicates of hiPSC-derived SFEBs, demonstrating experiment-to-experiment consistency. Neurite detection using HCI was applied to assess neurite morphology. MEA analysis showed that firing and burst rates in SFEBs decreased with blockade of NMDARs and AMPARs and increased with GABAR blockade. We also demonstrate effective combination of both MEA and HCI to analyze VGlut+ populations surrounding electrodes within MEAs. HCI-based (Ca2+) transient analysis revealed firing in individual cells surrounding active MEA electrodes. Comparison with existing methodsCurrent methods to generate neural organoids show high degrees of variability, and often require sectioning or special handling for analysis. The protocol outlined in this manuscript generates SFEBs with high degree of consistency making them amenable to complex assays combining HTS and electrophysiology allowing for an in-depth, unbiased analysis. ConclusionsSFEBs can be used in combination with HTS to compensate for experimental variability common in 3D cultures, while significantly decreasing processing speed, making this an efficient starting point for phenotypic drug screening.

Signalling mechanisms for ROS-induced TRPM2 mediated microglial cell activation

Microglial cell is a highly plastic cell in which it retracts its branched processes upon activation by structurally diverse molecules. Elevation of these molecules in the brain has been implicated in a diversity of diseases conditions in the CNS, where these molecules promote production of toxicity mediators, such as ROS. Microglial cell activation in response to ROS has been of particular interest. Emerging evidence supports a role for the TRPM2 channel in ROS-induced neuroinflammation. Thus, the current study aims to examine the role of the TRPM2 channel in mediating H2O2-induced microglial activation. A multidisciplinary approach was adopted, including primary microglial isolation, single cell calcium imaging, immunocytochemistry, confocal microscopy and computer-aided analysis of cell morphology. H2O2-induced microglial activation were observed in WT microglial cells but were ablated by genetic or pharmacological inhibition of the TRPM2 channel. Exposure to H2O2 raised the [Ca2+]i via promoting Ca2+ influx, which was prevented by TRPM2-KO. H2O2 induced ROS production and PARP-1 activation.  H2O2induced ROS production and PARP-1 activation as well as an increase in the [Ca2+]i and microglial activation, were suppressed by inhibiting PKC and NOX. Furthermore, H2O2-induced PARP-1 activation, increase in the [Ca2+]i and microglial activation were attenuated by inhibiting the Ca2+-sensitive PYK2 and downstream MEK/ERK kinases. The findings provide strong evidence to support that the TRPM2 channel is functionally expressed and plays a major role in ROS-induced Ca2+ signalling as well as cell activation in microglia. Such information is useful for a better understanding of microglial cells in oxidative stress-related pathologies. 

Neurotrophic Proteins in Dentin and Their Effect on Trigeminal Sensory Neurons

IntroductionA plethora of bioactive molecules present during tooth formation become sequestered in the mineralized dentin matrix and can be released into the pulp tissue after demineralization from carious lesions. However, neurotrophic factors are differentially expressed and secreted during various stages of odontogenesis. Thus, the aims of this study were(1) to investigate their presence and relative abundance in crown and root dentin and(2) to evaluate the bioactivity of dentin-derived proteins on neuronal cells. MethodsDentin matrix proteins (DMPs) were isolated from matched roots and crowns of extracted healthy human third molars. The total protein amount as well as the concentration of growth factors and neurotrophic proteins were quantified. The impact on neuritogenesis was determined with mouse trigeminal neurons in vitro and by a hydrogel implant model in vivo. Transient receptor potential cation channel subfamily V member 1 (TRPV1) sensitization of DMP-conditioned neurons was evaluated by single-cell calcium imaging. ResultsThe relative concentration of neurotrophic molecules revealed that nerve growth factor is the most abundant neurotrophin with 3-fold increased expression in radicular dentin. Similarly, brain-derived neurotrophic factor and neurotrophin 3 are more abundant in radicular than coronal dentin. Conversely, glial cell line–derived neurotrophic factor is more abundant in coronal dentin, whereas neurotrophin 4 is equally distributed. Dentin matrix proteins promoted neurite outgrowth in vitro and axonal targeting in vivo, with a greater effect observed by radicular dentin extracts. Furthermore, DMPs sensitized TRPV1 responses in mouse trigeminal neurons with greater activity seen with extracts from root dentin. ConclusionsNeurotrophic factors are differentially distributed between coronal and radicular dentin with different effects of dentin-derived proteins on axonal growth and targeting as well as the sensitization of TRPV1. Thus, extracellular proteins from the dentin matrix are likely involved in neurogenic responses to caries and could be exploited in clinical regenerative endodontics to promote reinnervation and enhance tissue regeneration.

Unique contributions of parvalbumin and cholinergic interneurons in organizing striatal networks during movement.

Striatal pavalbumin (PV) and cholinergic (CHI) interneurons are poised to play major roles in behavior by coordinating the networks of medium spiny cells that relay motor output. However, the small numbers and scattered distribution of these cells has made it difficult to directly assess their contribution to activity in networks of MSNs during behavior. Here, we build upon recent improvements in single cell calcium imaging combined with optogenetics to test the capacity of PVs and CHIs to affect MSN activity and behavior in mice engaged in voluntarily locomotion. We find that PVs and CHIs have unique effects on MSN activity and dissociable roles in supporting movement. PV cells facilitate movement by refining the activation of MSN networks responsible for movement execution. CHIs, in contrast, synchronize activity within MSN networks to signal the end of a movement bout. These results provide new insights into the striatal network activity that supports movement.

Long-Term Diabetic Microenvironment Augments the Decay Rate of Capsaicin-Induced Currents in Mouse Dorsal Root Ganglion Neurons.

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.

Cannabinoids affect differentiation of human iPSC-derived neurons

Event Abstract Back to Event Cannabinoids affect differentiation of human iPSC-derived neurons Tiago C. Barata1, Claudia Miranda1, Sandra Vaz2, Carla S. Ferreira3, Alexandre Quintas3 and Evguenia P. Bekman1, 2* 1 Institute for Biotechnology and Bioengineering, Portugal 2 Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal 3 Centro de investigação interdisciplinar Egas Moniz, Portugal Phytocannabinoids are psychotropic substances found in cannabis that bind to the endocannabinoid receptors regulating a variety of physiological processes in human body, including synaptic activity in the central nervous system and metabolic effects in the peripheric nervous system, among many others (1,2). Synthetic cannabinoids emerged as popular alternative to cannabis. Most of these substances are synthetic analogues of Δ9-THC, the psychotropic compound of cannabis, usually with higher affinity to the endocannabinoid receptor CB1 and/or a slower dissociation kinetics, capable to elicit a stronger and long-lasting effect on brain cells. Synthetic cannabinoids are easily available, and their molecular structure is always changing, increasing hazard for general population. The popularity of cannabis and its derivatives may lead, and often does, to child’s exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with higher risk of newborn morbidity (2,3), altered rate of mental development and significant changes in nervous system functioning (4,5). However, direct evidence that these effects are mediated through the binding of cannabinoids to endocannabinoid receptors is still lacking. Thus, it is paramount to better understand the psychoactive effects of natural and synthetic cannabinoids, on the developing human brain. We conveyed a pilot study in which human induced pluripotent stem cells (hiPSCs) were induced into neural differentiation and treated with a non-psychotropic component of cannabis, cannabinol, known to bind the CB2 receptor, and two synthetic Δ9-THC analogues, THJ and EG-018. The goal of this study was to test the hypothesis that exposure of developing human neural cell to cannabinoids influences neuronal differentiation. To approach this, hiPS cells were induced into in vitro neural commitment and differentiation using modified dual SMAD inhibition protocol (6) to allow for functional maturation and exposed from day 19 to day 32 of differentiation to EG-18, THJ and cannabinol directly supplemented to the culture medium. Cultures were analysed at days 32, 56 and 80 of differentiation for the presence of neural progenitor, neuronal and glial markers by immunofluorescence and additionally, functional analysis of the cultured cells was performed at day 56 by single cell calcium imaging (SCCI). The data revealed striking differences between untreated control and treated cells. Cultures treated with cannabinol exhibited a decrease in PAX6+ progenitors by day 32 and almost total depletion in GFAP+ progenitor/glial cells on later stages. MAP+ neuronal cells showed shorter non-ramified neurites and very few functional neurons capable to respond to KCl stimulus were found in this condition indicating a strong negative effect of this substance on neuronal differentiation. Contrary to the cannabinol, cultures treated with synthetic cannabinoids demonstrated presence of higher proportion of mature neurons in both cultures at day 56, indicating that both synthetic cannabinoids are able to promote neuronal differentiation also increase the length and branching of neurites in both MAP+ neurons and GFAP+ glia. However, SCCI analysis showed that the functionality of neural cells grown in these conditions appears to be compromised. Further studies are needed to better understand the observed functional impairment of cannabinoid-treated neurons. Overall these data clearly show that all three compounds used in this study strongly influence the process of neuronal differentiation and thus, most likely are able to impact on the development of human CNS. Acknowledgements Authors thank Cooperativa de Ensino Superior Egas Moniz, CRL for funding this research

Neuronal Activity-Dependent Activation of Astroglial Calcineurin in Mouse Primary Hippocampal Cultures.

Astrocytes respond to neuronal activity by generating calcium signals which are implicated in the regulation of astroglial housekeeping functions and/or in modulation of synaptic transmission. We hypothesized that activity-induced calcium signals in astrocytes may activate calcineurin (CaN), a calcium/calmodulin-regulated protein phosphatase, implicated in neuropathology, but whose role in astroglial physiology remains unclear. We used a lentiviral vector expressing NFAT-EYFP (NY) fluorescent calcineurin sensor and a chemical protocol of LTP induction (cLTP) to show that, in mixed neuron-astrocytic hippocampal cultures, cLTP induced robust NY translocation into astrocyte nuclei and, hence, CaN activation. NY translocation was abolished by the CaN inhibitor FK506, and was not observed in pure astroglial cultures. Using Fura-2 single cell calcium imaging, we found sustained Ca2+ elevations in juxtaneuronal, but not distal, astrocytes. Pharmacological analysis revealed that both the Ca2+ signals and the nuclear NY translocation in astrocytes required NMDA and mGluR5 receptors and depended on extracellular Ca2+ entry via a store-operated mechanism. Our results provide a proof of principle that calcineurin in astrocytes may be activated in response to neuronal activity, thereby delineating a framework for investigating the role of astroglial CaN in the physiology of central nervous system.

Behavioral Strategy Determines Frontal or Posterior Location of Short-Term Memory in Neocortex

The location of short-term memory in mammalian neocortex remains elusive. Here we show that distinct neocortical areas maintain short-term memory depending on behavioral strategy. Using wide-field and single-cell calcium imaging, we measured layer 2/3 neuronal activity in mice performing a whisker-based texture discrimination task with delayed response. Mice either deployed an active strategy-engaging their body toward the approaching texture-or passively awaited the touch. Independent of strategy, whisker-related posterior areas encoded choice early after touch. During the delay, in contrast, persistent cortical activity was located medio-frontally in active trials but in a lateral posterior area in passive trials. Perturbing these areas impaired performance for the associated strategy and also provoked strategy switches. Frontally maintained information related to future action, whereas activity in the posterior cortex reflected past stimulus identity. Thus, depending on behavioral strategy, cortical activity is routed differentially to hold information either frontally or posteriorly before converging to similar action.

Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina

Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l-Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.

The human bitter taste receptor T2R38 is broadly tuned for bacterial compounds

T2R38 has been shown to be a specific bacterial detector implicated in innate immune defense mechanism of human upper airway. Several clinical studies have demonstrated that this receptor is associated with the development of chronic rhinosinusitis (CRS). T2R38 was previously reported to bind to homoserine lactones (HSL), quorum sensing molecules specific of Pseudomonas Aeruginosa and other gram negative species. Nevertheless, these bacteria are not the major pathogens found in CRS. Here we report on the identification of bacterial metabolites acting as new agonists of T2R38 based on a single cell calcium imaging study. Two quorum sensing molecules (Agr D1 thiolactone from Staphylococcus Aureus and CSP-1 from Streptococcus Pneumoniae) and a list of 32 bacterial metabolites from pathogens frequently implicated in CRS were tested. First, we observed that HSL failed to activate T2R38 in our experimental system, but that the dimethylsulfoxide (DMSO), used as a solvent for these lactones may, by itself, account for the agonistic effect previously described. Secondly, we showed that both Agr D1 thiolactone and CSP-1 are inactive but that at least 7 bacterial metabolites (acetone, 2-butanone, 2-pentanone, 2-methylpropanal, dimethyl disulfide, methylmercaptan, γ-butyrolactone) are able to specifically trigger this receptor. T2R38 is thus much more broadly tuned for bacterial compounds than previously thought.

The anti-migraine component of butterbur extracts, isopetasin, desensitizes peptidergic nociceptors by acting on TRPA1 cation channel.

The mechanism of the anti-migraine action of extracts of butterbur [Petasites hybridus (L.) Gaertn.] is unknown. Here, we investigated the ability of isopetasin, a major constituent of these extracts, to specifically target TRPA1 channel and to affect functional responses relevant to migraine. Single-cell calcium imaging and patch-clamp recordings in human and rodent TRPA1-expressing cells, neurogenic motor responses in rodent isolated urinary bladder, release of CGRP from mouse spinal cord in vitro and facial rubbing in mice and meningeal blood flow in rats were examined. Isopetasin induced (i) calcium responses and currents in rat/mouse trigeminal ganglion (TG) neurons and in cells expressing the human TRPA1, (ii) substance P-mediated contractions of rat isolated urinary bladders and (iii) CGRP release from mouse dorsal spinal cord, responses that were selectively abolished by genetic deletion or pharmacological antagonism of TRPA1 channels. Pre-exposure to isopetasin produced marked desensitization of allyl isothiocyanate (AITC, TRPA1 channel agonist)- or capsaicin (TRPV1 channel agonist)-evoked currents in rat TG neurons, contractions of rat or mouse bladder and CGRP release from mouse central terminals of primary sensory neurons. Repeated intragastric administration of isopetasin attenuated mouse facial rubbing, evoked by local AITC or capsaicin, and dilation of rat meningeal arteries by acrolein or ethanol (TRPA1 and TRPV1 channel agonists respectively). Activation of TRPA1 channels by isopetasin results in excitation of neuropeptide-containing nociceptors, followed by marked heterologous neuronal desensitization. Such atten uation in pain and neurogenic inflammation may account for the anti-migraine action of butterbur.

ESTABLISHING MICROFLUIDIC DEVICES AS AN IN VITRO NEURONAL MODEL FOR STUDYING MOLECULAR AND FUNCTIONAL ASPECTS OF TRANSNEURONAL TAU PROPAGATION

A hallmark of neurodegenerative diseases, such as Alzheimer's disease and frontotemporal dementia, is aggregation and accumulation of tau protein as intracellular neurofibrillary tangles. Recent scientific evidence suggests the propagation of pathological tau across neuronal networks by a mechanism involving trans-synaptic release and reuptake of pathological tau aggregates. Herein, we characterised in vitro the molecular and functional aspects of neuronal tau pathology in a microfluidics cellular model that recapitulates the propagation of tau aggregation. Primary rat cortical neurons were cultured in microfluidic devices and transduced with viral vectors to express human tau proteins. Neurons were incubated with insoluble aggregated species of tau extracted from the P301S transgenic mice and analysed over various time intervals by means of staining with specific antibodies. Single-cell calcium imaging was employed to study network connectivity of the neurons in the microfluidic devices. By using selective synaptic modulators we have demonstrated synaptic propagation of evoked neuronal responses between different compartments of the microfluidic system. Subsequent to incubation with pathological P301S tau we have shown both direct seeding as well as neuron-to-neuron propagation of the aggregated tau, following a time and concentration dependence. We are also reporting here a novel image analysis procedure that was optimised for in depth quantification of immunolabeling data generated by using microfluidic devices. Overall, the present study established an in vitro system, in which neurons are synaptically connected, for studying trans-neuronal propagation of tau that would potentially assist in developing future therapeutic strategies to inhibit the spread of pathological tau.

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.