Native Neurons Research Articles

Modulation of dopamine's effect at dopamine 2 receptors (D2R) mediated by association with the ghrelin receptor, GHSR

The typical action of dopamine at the dopamine 2 receptor (D2R) on CNS neurons is inhibition, an effect mediated via Gαi/o through GIRK channels. However, at two sites, autonomic preganglionic neurons in the lumbosacral spinal cord, and in the lateral hypothalamus, dopamine causes neuronal excitation through D2R. At both sites, dopamine neurons that are excited by dopamine express the ghrelin receptor, GHSR. Ghrelin is absent from the lateral hypothalamus and spinal cord, and it has been proposed that GHSR may modulate D2R signaling by the formation of heterodimers.In lumbrosacral spinal cord neurons of the defecation centre, D2R and GHSR agonists applied successively were both excitatory. Antagonism of GHSR at these neurons blocked the excitatory effect of DR2 stimulation, which was also blocked by depletion of intracellular calcium (iCa2+). We further investigated this interaction in recombinant cultured cells. In CHO cells expressing D2R, dopamine agonists had almost no effect on iCa2+, whereas in the presence of GHSR, D2R coupling to iCa2+ was observed in response to nanomolar dopamine. The elevation of iCa2+ by dopamine in D2R/GHSR cells was reduced by either D2R or GHSR antagonism, but the effect of a GHSR agonist was reduced only by GHSR antagonism. D2R coupling to iCa2+ in the presence of GHSR was dependent on both Gαq and Gαi/o, whereas ghrelin agonist coupling was dependent only on Gαq. D2R mediated effects on cAMP were dependent only on Gαi/o and were not effected by GHSR antagonism. D2R and GHSR in the membranes of CHO cells, revealed by fluorescent ligands, moved independently when tracked at high resolution in real time and, using fluorescent lifetime imaging, individual labelled DR2 sites were not in close enough proximity to detect resonance energy transfer (no detectable FRET). Consistent with coupling being via downstream crosstalk, no detectable DR2 dependent iCa2+ was evident in cells expressing the GHSR – A204E mutant, which lacks constitutive activity. The DR2-dependent iCa2+ increase was restored in these cells using subthreshold pre-stimulation with ghrelin. In native neurons of the defecation center, inward currents in response to D2R agonism were blocked by U73122, an inhibitor of PLCβ. Together this data indicates that dopamine-mediated excitation was dependent on GHSR, PLCβ and iCa2+, in both native and recombinant cells. We conclude that co-expression of GHSR causes augmentation of agonist-induced, D2R-mediated PLCβ activation and store Ca2+ release in recombinant cells and in a population of autonomic preganglionic neurons. D2R mediated excitation in D2R/GHSR cells is both Gαi/o and Gαq/11 dependent, but does not require receptor dimerisation. Our findings reveal a novel interaction between GPCRs, in a physiologically relevant system, that does not require direct receptor interaction and has broad implications for recoding of metabotropic neurotransmitter responses via modulation through other GPCRs. NHMRC grant, APP2012657, to SGBF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transient Receptor Potential Vanilloid 1 Signaling Is Independent on Protein Kinase A Phosphorylation of Ankyrin-Rich Membrane Spanning Protein.

The sensory ion channel transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in small to medium sized dorsal root ganglion neurons, which are involved in the transfer of acute noxious thermal and chemical stimuli. The Ankyrin-rich membrane spanning protein (ARMS) interaction with TRPV1 is modulated by protein kinase A (PKA) mediating sensitization. Here, we hypothesize that PKA phosphorylation sites of ARMS are crucial for the modulation of TRPV1 function, and that the phosphorylation of ARMS is facilitated by the A-kinase anchoring protein 79 (AKAP79). We used transfected HEK293 cells, immunoprecipitation, calcium flux, and patch clamp experiments to investigate potential PKA phosphorylation sites in ARMS and in ARMS-related peptides. Additionally, experiments were done to discriminate between PKA and protein kinase D (PKD) phosphorylation. We found different interaction ratios for TRPV1 and ARMS mutants lacking PKA phosphorylation sites. The degree of TRPV1 sensitization by ARMS mutants is independent on PKA phosphorylation. AKAP79 was also involved in the TRPV1/ARMS/PKA signaling complex. These data show that ARMS is a PKA substrate via AKAP79 in the TRPV1 signaling complex and that all four proteins interact physically, regulating TRPV1 sensitization in transfected HEK293 cells. To assess the physiological and/or therapeutic significance of these findings, similar investigations need to be performed in native neurons and/or in vivo.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Discovery of 7-alkyloxy- [1,2,4] triazolo[1,5-a] pyrimidine derivatives as selective positive modulators of GABAA1 and GABAA4 receptors with potent antiepileptic activity

A series of 7-alkoxy - [1,2,4] triazolo [1, 5-a] pyrimidine derivatives were designed and synthesized. Maximal electroshock (MES) and pentylenetetrazole (PTZ) tests were utilized to access their anticonvulsant activity. Most of the series of compounds exhibited significant anti-seizure effects. Further studies demonstrated that the anticonvulsant activity of these compounds mainly depended on their allosteric potentiation of GABAA receptors. Among them, compound 10c was picked for the mechanism study due to its potent activity. The compound is more sensitive to subunit configurations of synaptic α1β2γ2 and extrasynaptic α4β3δ GABAA receptors, but there were no effects on NMDA receptors and Nav1.2 sodium channels. Meanwhile, 10c acted on the sites of GABAA receptors distinct from commonly used anticonvulsants benzodiazepines and barbiturates. Furthermore, studies from native neurons demonstrated that compound 10c also potentiated the activity of native GABAA receptors and reduced action potential firings in cultured cortical neurons. Such structural compounds may lay a foundation for further designing novel antiepileptic molecules.

An Electrophysiological and Pharmacological Study of the Properties of Human iPSC-Derived Neurons for Drug Discovery.

Human stem cell-derived neurons are increasingly considered powerful models in drug discovery and disease modeling, despite limited characterization of their molecular properties. Here, we have conducted a detailed study of the properties of a commercial human induced Pluripotent Stem Cell (iPSC)-derived neuron line, iCell [GABA] neurons, maintained for up to 3 months in vitro. We confirmed that iCell neurons display neurite outgrowth within 24 h of plating and label for the pan-neuronal marker, βIII tubulin within the first week. Our multi-electrode array (MEA) recordings clearly showed neurons generated spontaneous, spike-like activity within 2 days of plating, which peaked at one week, and rapidly decreased over the second week to remain at low levels up to one month. Extracellularly recorded spikes were reversibly inhibited by tetrodotoxin. Patch-clamp experiments showed that iCell neurons generated spontaneous action potentials and expressed voltage-gated Na and K channels with membrane capacitances, resistances and membrane potentials that are consistent with native neurons. Our single neuron recordings revealed that reduced spiking observed in the MEA after the first week results from development of a dominant inhibitory tone from GABAergic neuron circuit maturation. GABA evoked concentration-dependent currents that were inhibited by the convulsants, bicuculline and picrotoxin, and potentiated by the positive allosteric modulators, diazepam, chlordiazepoxide, phenobarbital, allopregnanolone and mefenamic acid, consistent with native neuronal GABAA receptors. We also show that glycine evoked robust concentration-dependent currents that were inhibited by the neurotoxin, strychnine. Glutamate, AMPA, Kainate and NMDA each evoked concentration-dependent currents in iCell neurons that were blocked by their selective antagonists, consistent with the expression of ionotropic glutamate receptors. The NMDA currents required the presence of the co-agonist glycine and were blocked in a highly voltage-dependent manner by Mg2+ consistent with the properties of native neuronal NMDA receptors. Together, our data suggest that such human iPSC-derived neurons may have significant value in drug discovery and development and may eventually largely replace the need for animal tissues in human biomedical research.

Epipregnanolone as a Positive Modulator of GABAA Receptor in Rat Cerebellar and Hippocampus Neurons.

Epipregnanolone (3β-hydroxy-5β-pregnan-20-one, Epi) is an endogenous steroid with important physiological effects and high affinity for GABAA receptors. The effect of Epi on GABA-induced chloride current (IGABA) in native neurons has hardly been studied. In this work, we studied the influence of Epi on the IGABA in the Purkinje cells of rat cerebellum and pyramidal neurons of rat hippocampus with the patch clamp technique. We showed that Epi is a positive modulator of the IGABA with EC50 of 5.7 µM in Purkinje cells and 9.3 µM in hippocampal neurons. Epi-induced potentiation of the IGABA was more potent at low vs. high GABA concentrations. Isopregnanolone (3β-hydroxy-5α-pregnan-20-one, Iso) counteracted Epi, reducing its potentiating effect by 2–2.3 times. Flumazenil, a nonsteroidal GABAA receptor antagonist, does not affect the Epi-induced potentiation. Comparison of the potentiating effects of Epi and allopregnanolone (3α-hydroxy-5α-pregnan-20-one, ALLO) showed that ALLO is, at least, a four times more potent positive modulator than Epi. The combined application of ALLO and Epi showed that the effects of these two steroids are not additive. We conclude that Epi has a dual effect on the IGABA increasing the current in the control solution and decreasing the stimulatory effect of ALLO.

How Can an Na+ Channel Inhibitor Ameliorate Seizures in Lennox-Gastaut Syndrome?

Lennox-Gastaut syndrome (LGS) is an epileptic encephalopathy frequently associated with multiple types of seizures. The classical Na+ channel inhibitors are in general ineffective against the seizures in LGS. Rufinamide is a new Na+ channel inhibitor, but approved for the treatment of LGS. This is not consistent with a choice of antiseizure drugs (ASDs) according to simplistic categorical grouping. The effect of rufinamide on the Na+ channel, cellular discharges, and seizure behaviors was quantitatively characterized in native neurons and mammalian models of epilepsy, and compared with the other Na+ channel inhibitors. With a much faster binding rate to the inactivated Na+ channel than phenytoin, rufinamide is distinctively effective if the seizure discharges chiefly involve short bursts interspersed with hyperpolarized interburst intervals, exemplified by spike and wave discharges (SWDs) on electroencephalograms. Consistently, rufinamide, but not phenytoin, suppresses SWD-associated seizures in pentylenetetrazol or AY-9944 models, which recapitulate the major electrophysiological and behavioral manifestations in typical and atypical absence seizures, including LGS. Na+ channel inhibitors shall have sufficiently fast binding to exert an action during the short bursts and then suppress SWDs, in which cases rufinamide is superior. For the epileptiform discharges where the interburst intervals are not so hyperpolarized, phenytoin could be better because of the higher affinity. Na+ channel inhibitors with different binding kinetics and affinity to the inactivated channels may have different antiseizure scope. A rational choice of ASDs according to in-depth molecular pharmacology and the attributes of ictal discharges is advisable. ANN NEUROL 2021;89:1099-1113.

Nucleated, Outside-Out, Somatic, Macropatch Recordings in Native Neurons.

Patch-clamp recordings are a powerful tool for the live measurement of the plasma membrane biophysical properties, with the ability to discriminate fast events such as fast inactivating Na+ currents (<1ms c.a.). It can be used in virtually every cell-type, including cardiomyocytes, skeletal muscles, neurons, and even epithelial cells and fibroblasts. Voltage-clamp, patch-clamp recordings can be used to measure and characterize the pharmacological and biophysical profile of membrane conductances, including leak, voltage-gated, and ligand-gated ion channels. This technique is particularly useful in studies carried out in cell-lines transfected with the gene expressing the conductance under investigation. However, voltage-clamp measures conducted on the soma of a native, adult neuron, for example in an acute brain slice or in the brain of a live individual, are subject to three major limitations: (1) the branching structure of the neuron causes space-clamp errors, (2) ion channels are differentially expressed across different neuronal compartments (such as soma, dendrites, and axons), and (3) the complex geometry of neurons makes it challenging to calculate current densities. While not preventing the experimenter to conduct patch-clamp, voltage-clamp recordings in native neurons, these limitations make the measures poorly standardized and hence often unusable for testing specific hypotheses.To overcome the limitations outlined above, outside-out, patch-clamp recordings can be carried out instead (See Chap. 1, Sect. 3.5); however, the signal-to-noise ratio in outside-outs from native, adult neurons is usually too low for obtaining accurate measurements.Here we describe how to carry out nucleated, outside-out, somatic, macropatch recordings (from now on abbreviated into "macropatch recordings") to obtain accurate and standardized measures of the biophysical and pharmacological properties of somatic, neuronal membrane conductances.

Building of AMPA-type glutamate receptors in the endoplasmic reticulum and its implication for excitatory neurotransmission.

AMPA-type glutamate receptors (AMPARs), the key elements of fast excitatory neurotransmission in the brain, are receptor ion channels whose core is assembled from pore-forming and three distinct types of auxiliary subunits. While it is well established that this assembly occurs in the endoplasmic reticulum (ER), it has remained largely enigmatic how this receptor-building happens. Here we review recent findings on the biogenesis of AMPARs in native neurons as a multistep production line that is defined and operated by distinct ER-resident helper proteins, and we discuss how impairment of these operators by mutations or targeted gene-inactivation leads to severe phenotypes in both humans and rodents. We suggest that the recent data on AMPAR biogenesis provide new insights into a process that is key to the formation and operation of excitatory synapses and their activity-dependent dynamics, as well as for the operation of the mammalian brain under normal and pathological conditions.

Immortalized Dorsal Root Ganglion Neuron Cell Lines.

Pain is one of the most significant causes of suffering and disability world-wide, and arguably the most burdensome global health challenge. The growing number of patients suffering from chronic pain conditions such as fibromyalgia, complex regional pain syndrome, migraine and irritable bowel syndrome, not only reflect the complexity and heterogeneity of pain types, but also our lack of understanding of the underlying mechanisms. Sensory neurons within the dorsal root ganglia (DRG) have emerged as viable targets for effective chronic pain therapy. However, DRG’s contain different classes of primary sensory neurons including pain-associated nociceptive neurons, non-nociceptive temperature sensing, mechanosensory and chemoreceptive neurons, as well as multiple types of immune and endothelial cells. This cell-population heterogeneity makes investigations of individual subgroups of DRG neurons, such as nociceptors, difficult. In attempts to overcome some of these difficulties, a limited number of immortalized DRG-derived cell lines have been generated over the past few decades. In vitro experiments using DRG-derived cell lines have been useful in understanding sensory neuron function. In addition to retaining phenotypic similarities to primary cultured DRG neurons, these cells offer greater suitability for high throughput assays due to ease of culture, maintenance, growth efficiency and cost-effectiveness. For accurate interpretation and translation of results it is critical, however, that phenotypic similarities and differences of DRG-derived cells lines are methodically compared to native neurons. Published reports to date show notable variability in how these DRG-derived cells are maintained and differentiated. Understanding the cellular and molecular differences stemming from different culture methods, is essential to validate past and future experiments, and enable these cells to be used to their full potential. This review describes currently available DRG-derived cell lines, their known sensory and nociceptor specific molecular profiles, and summarize their morphological features related to differentiation and neurite outgrowth.

Prolonged Amphetamine Treatments Cause Long-Term Decrease of Dopamine Uptake in Cultured Cells

Amphetamine (AMPH) is a systemic stimulant used to treat a variety of diseases including Attention Deficit Hyperactive Disorder, narcolepsy and obesity. Previous data showed that by binding to catecholamine transporters, AMPH prevents the reuptake of the neurotransmitters dopamine (DA) and norepinephrine (NE). Because AMPH, either used therapeutically at final concentrations of 1–10 µM or abused as recreational drug (50–200 µM), is taken over long periods of time, we investigated the prolonged effects of this drug on the uptake of DA. We found that, in LLC-PK1 cells stably expressing the human DA transporter (hDAT), pretreatments with 1 or 50 µM AMPH caused significant reduction in DA uptake right after the 15-h pretreatment. Remarkably, after 50 but not 1 µM AMPH pretreatment, we observed a significant reduction in DA uptake also after one, two or three cell divisions. To test whether these long-term effects induced by AMPH where conserved in a model comparable to primordial neuronal cells and native neurons, we used the human neuroblastoma cell line SH-SY5Y cells, which were reported to endogenously express both hDAT and the NE transporter. Pretreatments with 50 µM AMPH caused a significant reduction of DA uptake both right after 15 h and 3 cell divisions followed by neuro-differentiation with retinoic acid (RA) for 5 days. Under these same conditions, AMPH did not change the intracellular concentrations of ATP, ROS and cell viability suggesting, therefore, that the reduction in DA uptake was not cause by AMPH-induced toxicity. Interestingly, while 1 µM AMPH did not cause long-term effects in the LLC-PK1 cells, in the SH-SY5Y cells, it decreased the DA uptake after one, two, but not three, cell divisions and 5-day RA differentiation. These data show that besides the well-known acute effects, AMPH can also produce long-term effects in vitro that are maintained during cell division and transmitted to the daughter cells.

An ER Assembly Line of AMPA-Receptors Controls Excitatory Neurotransmission and Its Plasticity

Excitatory neurotransmission and its activity-dependent plasticity are largely determined by AMPA-receptors (AMPARs), ion channel complexes whose cell physiology is encoded by their interactome. Here, we delineate the assembly of AMPARs in the endoplasmic reticulum (ER) of native neurons as multi-state production line controlled by distinct interactome constituents: ABHD6 together with porcupine stabilizes pore-forming GluA monomers, and the intellectual-disability-related FRRS1l-CPT1c complexes promote GluA oligomerization and co-assembly of GluA tetramers with cornichon and transmembrane AMPA-regulatory proteins (TARP) to render receptor channels ready for ER exit. Disruption of the assembly line by FRRS1l deletion largely reduces AMPARs in the plasma membrane, impairs synapse formation, and abolishes activity-dependent synaptic plasticity, while FRRS1l overexpression has the opposite effect. As a consequence, FRSS1l knockout mice display severe deficits in learning tasks and behavior. Our results provide mechanistic insight into the stepwise biogenesis of AMPARs innative ER membranes and establish FRRS1l asapowerful regulator of synaptic signaling and plasticity.

The Cannabinoid-Like Compound, VSN16R, Acts on Large Conductance, Ca2+-Activated K+ Channels to Modulate Hippocampal CA1 Pyramidal Neuron Firing.

Large conductance, Ca2+-activated K+ (BKCa) channels are widely expressed in the central nervous system, where they regulate action potential duration, firing frequency and consequential neurotransmitter release. Moreover, drug action on, mutations to, or changes in expression levels of BKCa can modulate neuronal hyperexcitability. Amongst other potential mechanisms of action, cannabinoid compounds have recently been reported to activate BKCa channels. Here, we examined the effects of the cannabinoid-like compound (R,Z)-3-(6-(dimethylamino)-6-oxohex-1-en-1-yl)-N-(1-hydroxypropan-2-yl) benzamide (VSN16R) at CA1 pyramidal neurons in hippocampal ex vivo brain slices using current clamp electrophysiology. We also investigated effects of the BKCa channel blockers iberiotoxin (IBTX) and the novel 7-pra-martentoxin (7-Pra-MarTx) on VSN16R action. VSN16R (100 μM) increased first and second fast after-hyperpolarization (fAHP) amplitude, decreased first and second inter spike interval (ISI) and shortened first action potential (AP) width under high frequency stimulation protocols in mouse hippocampal pyramidal neurons. IBTX (100 nM) decreased first fAHP amplitude, increased second ISI and broadened first and second AP width under high frequency stimulation protocols; IBTX also broadened first and second AP width under low frequency stimulation protocols. IBTX blocked effects of VSN16R on fAHP amplitude and ISI. 7-Pra-MarTx (100 nM) had no significant effects on fAHP amplitude and ISI but, unlike IBTX, shortened first and second AP width under high frequency stimulation protocols; 7-Pra-MarTx also shortened second AP width under low frequency stimulation protocols. However, in the presence of 7-Pra-MarTx, VSN16R retained some effects on AP waveform under high frequency stimulation protocols; moreover, VSN16R effects were revealed under low frequency stimulation protocols. These findings demonstrate that VSN16R has effects in native hippocampal neurons consistent with its causing an increase in initial firing frequency via activation of IBTX-sensitive BKCa channels. The differential pharmacological effects described suggest that VSN16R may differentially target BKCa channel subtypes.

Role of Kv7.2/Kv7.3 and M1 muscarinic receptors in the regulation of neuronal excitability in hiPSC-derived neurons

The Kv7 family of voltage-dependent non-inactivating potassium channels is composed of five members, of which four are expressed in the CNS. Kv7.2, 7.3 and 7.5 are responsible for the M-current, which plays a critical role in the regulation of neuronal excitability. Stimulation of M1 muscarinic acetylcholine receptor, M1 receptor, increases neuronal excitability by suppressing the M-current generated by the Kv7 channel family. The M-current modulation via M1 receptor is well-described in in vitro assays using cell lines and in native rodent tissue. However, this mechanism was not yet reported in human induced pluripotent stem cells (hiPSC) derived neurons. In the present study, we investigated the effects of both agonists and antagonists of Kv7.2/7.3 channel and M1 receptor in hiPSC derived neurons and in primary rat cortical neuronal cells. The role of M1 receptors in the modulation of neuronal excitability could be demonstrated in both rat primary and hiPSC neurons. The M1 receptors agonist, xanomeline, increased neuronal excitability in both rat cortical and the hiPSC neuronal cells. Furthermore, M1 receptor agonist-induced neuronal excitability in vitro was reduced by an agonist of Kv7.2/7.3 in both neuronal cells. These results show that hiPSC derived neurons recreate the modulation of the M-current by the muscarinic receptor in hiPSC neurons similarly to rat native neurons. Thus, hiPSC neurons could be a useful human-based cell assay for characterization of drugs that affect neuronal excitability and/or induce seizure activity by modulation of M1 receptors or inhibition of Kv7 channels.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

Epigenetic Analysis in Human Neurons: Considerations for Disease Modeling in PD.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease. Most PD cases are considered to be sporadic and despite considerable scientific effort, the underlying cause(s) still remain(s) enigmatic. In particular, it is unknown to which extent epigenetic alterations contribute to the pathophysiology of this devastating disorder. This is partly due to the fact that appropriate PD models are not yet available. Moreover, epigenetic patterns and mechanisms are species specific and murine systems reflect only a few of the idiosyncrasies of human neurons. For several years now, patient-specific stem cell-derived neural and non-neural cells have been employed to overcome this limitation allowing the analysis and establishment of humanized disease models for PD. Thus, several studies tried to dissect epigenetic alterations such as aberrant DNA methylation or microRNA patterns using lund human mesencephalic cell lines or neurons derived from (patient-specific) induced pluripotent stem cells. These studies demonstrate that human neurons have the potential to be used as model systems for the study of epigenetic modifications in PD such as characterizing epigenetic changes, correlating epigenetic changes to gene expression alterations and hopefully using these insights for the development of novel therapeutics. However, more research is required to define the epigenetic (age-associated) landscape of human in vitro neurons and compare these to native neurons before they can be established as suitable models for epigenetic studies in PD. In this review, we summarize the knowledge about epigenetic studies performed on human neuronal PD models, and we discuss advantages and current limitations of these (stem cell-derived) neuronal models for the study of epigenetic alterations in PD.

Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons

SummaryG protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs.

Unmasking GluN1/GluN3A excitatory glycine NMDA receptors

GluN3A and GluN3B are glycine-binding subunits belonging to the NMDA receptor (NMDAR) family that can assemble with the GluN1 subunit to form unconventional receptors activated by glycine alone. Functional characterization of GluN1/GluN3 NMDARs has been difficult. Here, we uncover two modalities that have transformative properties on GluN1/GluN3A receptors. First, we identify a compound, CGP-78608, which greatly enhances GluN1/GluN3A responses, converting small and rapidly desensitizing currents into large and stable responses. Second, we show that an endogenous GluN3A disulfide bond endows GluN1/GluN3A receptors with distinct redox modulation, profoundly affecting agonist sensitivity and gating kinetics. Under reducing conditions, ambient glycine is sufficient to generate tonic receptor activation. Finally, using CGP-78608 on P8-P12 mouse hippocampal slices, we demonstrate that excitatory glycine GluN1/GluN3A NMDARs are functionally expressed in native neurons, at least in the juvenile brain. Our work opens new perspectives on the exploration of excitatory glycine receptors in brain function and development.

Direct Reprogramming of Astrocytes into Functional Serotonergic Neurons

Serotonergic neurons project widely in the central nervous system and regulate a variety of behaviors. Obtaining induced serotonergic neurons (iSNs) iscritical for disease modeling, drug screening and cell therapy. Here, we found that a combination of transcription factors Ascl1, Nkx2.2, Gata2, Foxa2, and Lmx1b could convert postnatal astrocytes into functional serotonergic neurons. We further found that the iSNs expressed serotonergic neuron-specific markers and exhibited similar electrophysiological properties with native serotonergic neurons. More importantly, using an optogenetic stimulation protocol, we demonstrated that the iSNs could form synaptic connections with co-cultured neurons in dishes. Last, after transplanted into the mouse brain, astrocyte-derived iSNs survived, expressed appropriate molecular markers, and matured gradually in vivo. Taken together, our results showed that defined transcription factors were able to convert astrocytes into functional serotonergic neurons andthus provided an alternative option for generate sufficient numbers of a new neuronal subtype for therapeutic therapy.

Potent block of potassium channels by MEK inhibitor U0126 in primary cultures and brain slices

U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), a widely used mitogen-activated protein kinase kinase (MEK) inhibitor, was found to accelerate voltage-gated K+ channel (KV) inactivation in heterologous cells expressing several types of KV. The goal of this study was to examine whether U0126 at a concentration thought to specifically inhibit MEK signaling also inhibits KV in native neurons of primary cultures or brain slices. U0126 caused a dose-dependent inhibition of both the transient (IA) and sustained (IDR) components of K+ currents in hippocampal neurons. U0126 also exhibited much higher potency on the IA and IDR than the classical KV blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Consistent with its inhibitory effect on KV, U0126 broadened action potential duration, profoundly affected the repolarizing phase, and dramatically reduced firing frequency in response to current pulse injections. Despite the potent and reversible action of U0126 on Kv channels, PD98059, a structurally-unrelated MEK inhibitor, did not induce such an effect, suggesting U0126 may act independently of MEK inhibition. Together, these results raise cautions for using U0126 as a specific inhibitor for studying MEK signaling in neurons; on the other hand, further studies on the blocking mechanisms of U0126 as a potent inhibitor of KV may provide useful insights into the structure-function relationship of KV in general.