Fenglian Xu

Mercury-induced toxicity of rat cortical neurons is mediated through N-methyl-D-Aspartate receptors

BackgroundMercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer’s disease, Parkinson’s disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid) on cultured, rat cortical neurons.ResultsWe found that inorganic mercuric chloride (HgCl2 –at 0.025 to 25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Whole-cell patch-clamp recordings of pyramidal neurons revealed that HgCl2 not only enhanced the amplitude and frequency of synaptic, inward currents, but also increased spontaneous synaptic potentials followed by sustained membrane depolarization. HgCl2 also triggered sustained, 2–5 fold rises in intracellular calcium concentration ([Ca2+]i). The observed increases in neuronal activity and [Ca2+]i were substantially reduced by the application of MK 801, a non-competitive antagonist of N-Methyl-D-Aspartate (NMDA) receptors. Importantly, our study further shows that a pre incubation or co-application of MK 801 prevents HgCl2-induced reduction of cell viability and a disruption of β-tubulin.ConclusionsCollectively, our data show that HgCl2-induced toxic effects on central neurons are triggered by an over-activation of NMDA receptors, leading to cytoskeleton instability.

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca2+ concentration ([Ca2+]i) of type I cells dissociated from rat carotid bodies using a Ca2+ imaging technique. ATP did not affect the resting [Ca2+]i but strongly suppressed the hypoxia‐induced [Ca2+]i elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2‐methylthioATP > ATP > ADP ≫ α, β‐methylene ATP > UTP, implicating the involvement of P2Y1 receptors. Simultaneous measurements of membrane potential and [Ca2+]i show that ATP inhibited the hypoxia‐induced Ca2+ signal by reversing the hypoxia‐triggered depolarization. However, ATP did not oppose the hypoxia‐mediated inhibition of the oxygen‐sensitive TASK‐like K+ background current. Neither the inhibition of the large‐conductance Ca2+‐activated K+ (maxi‐K) channels nor the removal of extracellular Na+ could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK‐like K+, maxi‐K or Na+ channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the oxygen sensing type I (glomus) cells of rat carotid bodies via reduction of a background TASK-like K+ current

Pituitary adenylate cyclase‐activating polypeptide (PACAP)‐deficient mice are prone to sudden neonatal death and have reduced respiratory response to hypoxia. Here we found that PACAP‐38 elevated cytosolic [Ca2+] ([Ca2+]i) in the oxygen sensing type I cells but not the glial‐like type II (sustentacular) cells of the rat carotid body. This action of PACAP could not be mimicked by vasoactive intestinal peptide but was abolished by PACAP 6‐38, implicating the involvement of PAC1 receptors. H89, a protein kinase A (PKA) inhibitor attenuated the PACAP response. Simultaneous measurement of membrane potential and [Ca2+]i showed that the PACAP‐mediated [Ca2+]i rise was accompanied by depolarization and action potential firing. Ni2+, a blocker of voltage‐gated Ca2+ channels (VGCC) or the removal of extracellular Ca2+ reversibly inhibited the PACAP‐mediated [Ca2+]i rise. In the presence of tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP), PACAP reduced a background K+ current. Anandamide, a blocker of TWIK‐related acid‐sensitive K+ (TASK)‐like K+ channel, occluded the inhibitory action of PACAP on K+ current. We conclude that PACAP, acting via the PAC1 receptors coupled PKA pathway inhibits a TASK‐like K+ current and causes depolarization and VGCC activation. This stimulatory action of PACAP in carotid type I cells can partly account for the role of PACAP in respiratory disorders.

Trophic Factor-Induced Intracellular Calcium Oscillations Are Required for the Expression of Postsynaptic Acetylcholine Receptors during Synapse Formation between Lymnaea Neurons

Nervous system functions in all animals rely upon synaptic connectivity that is established during early development. Whereas cell–cell signaling plays a critical role in establishing synapse specificity, the involvement of extrinsic growth factors cannot, however, be undermined. We have previously demonstrated that trophic factors are required for excitatory but not inhibitory synapse formation between Lymnaea neurons. Moreover, in the absence of trophic factors, neurons from a number of species establish inappropriate inhibitory synapses, which can, however, be corrected by the addition of trophic factors. The precise site of trophic factor actions (presynaptic versus postsynaptic) and the underlying mechanisms remain, however, undefined. Here, we provide the first direct evidence that the trophic factor-mediated excitatory synapse formation involves activity-induced calcium (Ca2+) oscillations in the postsynaptic left pedal dorsal 1 (LPeD1) but not the presynaptic visceral dorsal 4 (VD4, cholinergic) neuron. These oscillations involved Ca2+ influx through voltage-gated Ca2+ channels and required receptor tyrosine kinase activity which was essential for the expression of excitatory, nicotinic acetylcholine receptors in the postsynaptic cell during synapse formation. We also demonstrate that selectively blocking the electrical activity presynaptically did not perturb trophic factor-induced synapse formation between the paired cells, whereas hyperpolarizing the postsynaptic cell prevented appropriate synaptogenesis between VD4 and LPeD1 cells. Together, our data underscore the importance of extrinsic trophic factors in regulating the electrical activity of the postsynaptic but not the presynaptic cell and that the resulting Ca2+ oscillations are essential for the expression of postsynaptic receptors during specific synapse formation.

Quercetin Targets Cysteine String Protein (CSPα) and Impairs Synaptic Transmission

Background Cysteine string protein (CSPα) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPα is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPα in mice results in knockout mice that are normal for the first 2–3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPα prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPα represents a promising therapeutic target for the prevention of neurodegenerative disorders. Methodology/Principal Findings Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPα-CSPα dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPα dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPα function. Quercetins action on CSPα is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPα:Hsc70 units (70kDa heat shock cognate protein). Conclusions/Significance Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPα and the undesired consequences of CSPα dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPα.

Antidepressant fluoxetine suppresses neuronal growth from both vertebrate and invertebrate neurons and perturbs synapse formation between Lymnaea neurons

Current treatment regimes for a variety of mental disorders involve various selective serotonin reuptake inhibitors such as Fluoxetine (Prozac). Although these drugs may ‘manage’ the patient better, there has not been a significant change in the treatment paradigm over the years and neither have the outcomes improved. There is also considerable debate as to the effectiveness of various selective serotonin reuptake inhibitors and their potential side‐effects on neuronal architecture and function. In this study, using mammalian cortical neurons, a dorsal root ganglia cell line (F11 cells) and identified Lymnaea stagnalis neurons, we provide the first direct and unequivocal evidence that clinically relevant concentrations of Fluoxetine induce growth cone collapse and neurite retraction of both serotonergic and non‐serotonergic neurons alike in a dose‐dependent manner. Using intracellular recordings and calcium imaging techniques, we further demonstrate that the mechanism underlying Fluoxetine‐induced effects on neurite retraction from Lymnaea neurons may involve lowering of intracellular calcium and a subsequent retardation of growth cone cytoskeleton. Using soma–soma synapses between identified presynaptic and postsynaptic Lymnaea neurons, we provide further direct evidence that clinically used concentrations of Fluoxetine also block synaptic transmission and synapse formation between cholinergic neurons. Our study raises alarms over potentially devastating side‐effects of this antidepressant drug on neurite outgrowth and synapse formation in a developing/regenerating brain. Our data also demonstrate that drugs such as Fluoxetine may not just affect communication between serotonergic neurons but that the detrimental effects are widespread and involve neurons of various phenotypes from both vertebrate and invertebrate species.

Persistent sodium currents contribute to Aβ1-42-induced hyperexcitation of hippocampal CA1 pyramidal neurons

Patients with Alzheimers disease (AD) have elevated incidence of epilepsy. Moreover, neuronal hyperexcitation occurs in transgenic mouse models overexpressing amyloid precursor protein and its pathogenic product, amyloid β protein (Aβ). However, the cellular mechanisms of how Aβ causes neuronal hyperexcitation are largely unknown. We hypothesize that the persistent sodium current (INaP), a subthreshold sodium current that can increase neuronal excitability, may in part account for the Aβ-induced neuronal hyperexcitation. The present study was designed to evaluate the involvement of INaP in Aβ-induced hyperexcitation of hippocampal CA1 pyramidal neurons using a whole-cell patch-clamp recording technique. Our results showed that bath application of soluble Aβ1-42 increased neuronal excitability in a concentration-dependent manner. Soluble Aβ1-42 also increased the amplitude of INaP without significantly affecting its activation properties. In the presence of riluzole (RLZ), an antagonist of INaP, the Aβ1-42-induced neuronal hyperexcitation and INaP augmentation were significantly inhibited. These findings suggest that soluble Aβ1-42 may induce neuronal hyperexcitation by increasing the amplitude of INaP and that RLZ can inhibit the Aβ1-42-induced abnormal neuronal activity.

Gastrodin Suppresses the Amyloid β-Induced Increase of Spontaneous Discharge in the Entorhinal Cortex of Rats

Accumulated soluble amyloid beta- (Aβ-) induced aberrant neuronal network activity may directly contribute to cognitive deficits, which are the most outstanding characteristics of Alzheimers disease (AD). The entorhinal cortex (EC) is one of the earliest affected brain regions in AD. Impairments of EC neurons are responsible for the cognitive deficits in AD. However, little effort has been made to investigate the effects of soluble Aβ on the discharge properties of EC neurons in vivo. The present study was designed to examine the effects of soluble Aβ 1−42 on the discharge properties of EC neurons, using in vivo extracellular single unit recordings. The protective effects of gastrodin (GAS) were also investigated against Aβ 1−42-induced alterations in EC neuronal activities. The results showed that the spontaneous discharge of EC neurons was increased by local application of soluble Aβ 1−42 and that GAS can effectively reverse Aβ 1−42-induced facilitation of spontaneous discharge in a concentration-dependent manner. Moreover, whole-cell patch clamp results indicated that the protective function of GAS on abnormal hyperexcitability may be partially mediated by its inhibitory action on Aβ 1−42-elicited inward currents in EC neurons. Our study suggested that GAS may provide neuroprotective effects on Aβ 1−42-induced hyperactivity in EC neurons of rats.

The mitochondrial division inhibitor Mdivi-1 rescues mammalian neurons from anesthetic-induced cytotoxicity

BackgroundConcerns have risen regarding the potential side effects of clinical exposure of the pediatric population to inhalational anesthetics, and how they might impact cognitive, learning, and memory functions. However, neither the mechanisms of anesthetic cytotoxicity, nor potential protective strategies, have yet been fully explored. In this study, we examined whether two of the most commonly used inhalational anesthetics, sevoflurane and desflurane, affect neuronal viability and synaptic network assembly between cultured rat cortical neurons.ResultsPrimary rat cortical neuron cultures were exposed to equipotent sevoflurane or desflurane for 1 hour. Neuron viability, synaptic protein expression, mitochondrial morphology, and neurite growth were assayed with immunostaining and confocal microscopy techniques. The effects of anesthetics on the functional development of neural networks were evaluated with whole-cell patch clamp recordings of spontaneous synaptic currents. Our results demonstrate that an acute exposure to sevoflurane and desflurane inhibits the development of neurite processes, impacts the mitochondria, and compromises synaptic proteins - concomitant with a reduction in synaptic function in mature networks. Interestingly, pretreatment of neurons with a mitochondrial division inhibitor (Mdivi-1) not only protected mitochondria integrity but also played a protective role against anesthetic-induced structural and functional neurotoxicity.ConclusionsWe show that Mdivi-1 likely plays a protective role against certain harmful effects of general anesthetics on primary rat neuronal cultures. In addition, Mdivi-1 alone plays a direct role in enhancing growth and modulating synaptic activity. This study highlights the importance of further study into possible protective agents against anesthetic neurotoxicity.

Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments.