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In this work we quantified the in vitro calibration relationships between high frequency electrical stimulation and GABA and glutamate release in both mature retinoic acid differentiated P19 neurons and immortalized embryonic cortical cells engineered to express glutamic acid decarboxylase, GAD65. Extracellular glutamate and GABA was quantified by 2D gas chromatography and time of flight mass spectrometry after stimulation at varying amplitudes and frequencies. Amplitude sweeps resulted in a linear calibration for P19 neurons; the level of neurotransmitter varied over one order of magnitude from ~ 200 pg/neuron to ~ 1.2 ng/neuron for glutamate and ~ 1 ng/neuron to ~ 2 ng/neuron for GABA, depending on the stimulation amplitude. Frequency sweeps resulted in a peak release at 250 Hz for glutamate and 400 Hz for GABA in P19 cells. The GABA transporter inhibitor, nipecotic acid, increased extracellular GABA levels and decrease glutamate. In contrast the embryonic cortical cells had a strongly nonlinear dependency of release on stimulation amplitude, and a weak dependence on frequency. These cells had roughly equal extracellular glutamate and GABA levels after stimulation despite the expression of GAD65. In addition glutamate and GABA levels were insensitive to nipecotic acid. These results demonstrate an ability to calibrate and tune neurotransmitter release from neural cells using high frequency stimulation parameters.

Based on established physiological mechanisms, the paper presents a detailed computer model, which supports the hypothesis that temporal lobe epilepsy may be caused by failure of glutamate reuptake from the extracellular space. The elevated glutamate concentration causes an increased activation of NMDA receptors in pyramidal neurons, which in turn leads to neuronal dynamics that is qualitatively identical to epileptiform activity. We identify by chaos analysis a surprising possibility that muscarinergic receptors can help the system out of a chaotic regime.

Although many studies have indicated that electroacupuncture (EA) provides a neuroprotective effect against ischemic brain damage, the protective mechanism is not fully understood. Glutamate release and hippocampal blood flow in ischemia with EA were investigated to elucidate the neuroprotective mechanism of EA. Transient 5-minute ischemia was induced in gerbils. EA (7 Hz, 6 mA, for 30 minutes) delivered to the points called Fengfu (GV16) and Shendao (GV11) was administered pre-, intra- or post-ischemia. The procedure rescued hippocampal neurons from ischemic insult and significantly attenuated both ischemia-induced glutamate release and transient increase of cerebral blood flow (CBF) during reperfusion (hyperemia). Hyperemia as well as excessive glutamate after ischemia are regarded as important factors in brain damage as they lead to reperfusion injury. These results suggest that EA protects neurons by suppressing both glutamate release and reperfusion injury after ischemia.

Picroside II is an active constituent extracted from the traditional Chinese medicine (TCM) Hu-Huang-Lian. To evaluate the neuroprotective effect of picroside II, PC12 cells were treated with glutamate in vitro and male ICR mice were treated with AlCl₃in vivo. Pre-treatment of PC12 cells with picroside II could enhance the cell viability and decrease the level of intracellular reactive oxygen species (ROS) induced by glutamate. By DNA fragmentation and flow cytometry assay, picroside II (1.2 mg/ml) significantly prevented glutamate-induced cell apoptosis. In the animal study, amnesia was induced in mice by AlCl₃ (100 mg/kg/d, i.v.). Pricroside II, at the dose of 20 and 40 mg/kg/d (i.g.), markedly ameliorated AlCl₃-induced learning and memory dysfunctions and attenuated AlCl₃-induced histological changes. This was associated with the significant increased superoxide dismutase (SOD) activity in the brain of experimental mice. All these results indicated that picroside II possessed the therapeutic potential in protecting against neurological injuries damaged by oxidative stress.

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum (P. falciparum) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from $P.f$. 3D7 in vitro but also from Plasmodium berghei (P. berghei) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P. falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the “feedback pattern” is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

In this paper, a tripartite synapse network is constructed to examine external and internal triggering factors of epilepsy transition and propagation in neurons with the Epileptor-2 model. We first explore the external stimuli in the environment that induce epileptic activities and transition behaviors among Ictal Discharges (IDs) and Interictal Discharges (IIDs) states. The higher the strength and abruptness of the stimuli, the more severe is the occurrence of epilepsy within a reasonable range of parameters. Then for the internal triggering factors, the results of the tripartite synapse network, which is improved by combining the Epileptor-2 model with astrocyte by means of ion exchange and new connections, show that astrocytes can transmit normal physiological activity information and filter out abnormal discharge information of neurons. One of the causes for epileptic seizures is the abnormal release of glial neurotransmitters in astrocytes. The excessive release of glutamate causes the discharge state of neurons to transit from nonepileptic to IIDs, IDs and tonic, while adenosine triphosphate can alleviate epilepsy. Meanwhile, the synapse dysfunction of an astrocyte-free network can also lead to seizures, and the epilepsy propagation ability of a tripartite synapse network becomes weaker than that of an astrocyte-free network. Our research is expected to provide some theoretical basis for the therapeutic approach to curing epilepsy in the intracellular and extracellular contexts.

The role of cotransmission by α-amino-3-hydroxy-5-methyl-4-isoxalose propionic acid (AMPA), L-aspartate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) as well as the coexpression of AMPA, NMDA, and nicotinic ACh (nACh) receptors on the electrophysiological activity of the primary sensory (AH) and motor (S) neurons of the enteric nervous system are numerically assessed. Results of computer simulations showed that AMPA and L-Asp alone can induce fast action potentials of short duration on AH and S neurons. Costimulation of nACh and AMPA receptors on the soma of the S neuron resulted in periodic spiking activity. A characteristic biphasic response was recorded from the AH neuron after coactivation of AMPA and NMDA receptors. Glutamate alone acting on NMDA receptors caused prolonged depolarization of the AH neuron and failed to depolarize the S neuron. Cojoint stimulation of the AMPA or nACh receptors was required to produce the effect of glutamate. The overall electrical response of neurons to the activation of NMDA receptors was long-term depolarization. Acetylcholine, AMPA, and glutamate acting alone or cojointly enhanced phasic contraction of the longitudinal smooth muscle. Treatment of neurons with AMPA, NMDA, and nACh receptor antagonists revealed intricate properties of the AH and S neurons. Application of MK-801, D-AP5, and CPP reduced the excitability of the AH neuron and totally abolished electrical activity in the S neuron. The information gained into the cotransmission by excitatory amino acids and acetylcholine in the enteric nervous system may be beneficial in the development of novel effective therapeutics to treat diseases associated with altered visceral nociception, i.e. irritable bowel syndrome.

As discussed in other articles in this issue, chemical emergence may have led to the appearance of life on the pre-biotic earth, but it is even more obviously clear that emergence continues in living systems, producing complex phenomena such as ordering, biorhythms and even, possibly, consciousness. The role of continuing emergence in living systems is reviewed here with special attention to the Peroxidase–Oxidase reaction and neurochemical systems. For the latter, we review the role of subnetwork dynamics in epilepsy and an intriguing new possiblity that calcium waves in fields of astrocytes in the brain may be involved in the spread of epileptic seizures.

Recently, upregulation of metabotropic glutamate receptors (mGluRs) on hippocampal astrocytes in epileptic tissues has become part of the etiology of epilepsy and suggests the involvement of astrocytes in the disease. Through computational modeling, we have shown in previous work that upregulated mGluRs on astrocytes can give rise to positive feedback in closed loop neuron-astrocyte circuits with epilepsy-type spontaneous neuronal spiking. In this paper we further quantify the necessary degree of upregulation of astrocytic mGluRs, relate it to recent clinical and experimental studies, and address through computational modeling the role of synaptic inhibition through interneurons in this form of hyperexcitability. We conclude that inhibitive circuitry cannot tame this form of hyperexcitability.

Long-lasting, activity-dependent changes in synaptic efficacy at excitatory synapses are critical for experience-dependent synaptic plasticity. Synaptic plasticity at excitatory synapses is determined both presynaptically by changes in the probability of neurotransmitter release, and postsynaptically by changes in the availability of functional postsynaptic glutamate receptors. Two kinds of synaptic plasticity have been described. In homosynaptic or Hebbian plasticity, the events responsible for synaptic strengthening occur at the same synapse as is being strengthened. Homosynaptic plasticity is activity-dependent and associative, because it associates the firing of a postsynaptic neuron with that of the presynaptic neuron. Heterosynaptic plasticity, on the other hand, is activity-independent and the synaptic strength is modified as a result of the firing of a third, modulatory neuron. It has been suggested that long-term changes in synaptic strength, which are associated with gene transcription, can only be induced with the involvement of heterosynaptic plasticity. The neuromodulator adenosine plays an elaborated pre- and postsynaptic control of glutamatergic neurotransmission. This paper reviews the evidence suggesting that in some striatal excitatory synapses, adenosine can provide the heterosynaptic-like modulation essential for stabilizing homosynaptic plasticity without the need of a "third, modulatory neuron".

This commentary article extends Vimal's [J Integr Neurosci 7:49–73, 2008] concept of protoexperience by outlining a two-factor approach to the localization of consciousness within the physical matter of the brain consistent with contemporary theoretical physics, molecular and system biology, and neuroscience. Specific hypotheses based on this approach predict on clearly stated grounds the occurrence or non-occurrence, and degrees of intensity of consciousness within the human brain and possibly in related species based on the combination of protoconsciousness with energetic activating agents. In this it comprises a mechanics of consciousness.

The hippocampal formation is critically involved for the long-term storage of various forms of information, and it is widely believed that the phenomenon of long-term potentiation (LTP) of synaptic transmission is a molecular/cellular mechanism participating in memory formation. Although several high level models of hippocampal function have been developed, they do not incorporate detailed molecular information of the type necessary to understand the contribution of individual molecular events to the mechanisms underlying LTP and learning and memory. We are therefore developing new technological tools based on mathematical modeling and computer simulation of the molecular processes taking place in realistic biological networks to reach such an understanding. This article briefly summarizes the approach we are using and illustrates it by presenting data regarding the effects of changing the number of AMPA receptors on various features of glutamatergic transmission, including NMDA receptor-mediated responses and paired-pulse facilitation. We conclude by discussing the significance of these results and providing some ideas for future directions with this approach.

Photodynamic therapy based on photogeneration of cytotoxic singlet oxygen and following oxidative stress is currently used in neuro-oncology for destruction of brain tumors. However, along with a tumor, it damages healthy neurons and glial cells. We studied the involvement of the glutamate-related signaling pathway in photodynamic damage to normal glial cells in the crayfish stretch receptor. This model object consists of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens and irradiated by the diode laser (670 nm). Application of enzyme inhibitors and ion channels modulators showed that exogenous L-glutamate decreased photoinduced apoptosis of crayfish glial cells. The natural neuroglial mediator N-acetylaspartylglutamate, which releases glutamate after splitting by glutamate carboxypeptidase II, also inhibited photoinduced apoptosis. Inhibition of glutamate carboxypeptidase II, oppositely, enhanced glial apoptosis. This confirmed the antiapoptotic activity of glutamate. Glutamate agonist NMDA or inhibitor of NMDA receptors MK801 did not influence photodynamic death of glial cells, i.e., these receptors did not participate in glial apoptosis. Inhibition of metabotropic glutamate receptors mGluRI with AP-3 reduced PDT-induced apoptosis of glial cells. Thus, chemical modifiers of various signaling processes can modulate photoinduced necrosis or apoptosis of glial cells and thus modify efficiency of photodynamic therapy.

Injections of iron salts into the sensorimotor cortex, hippocampus, and amygdala of experimental animals results in chronic recurrent focal paroxysmal electroencephalographic discharges, behavioral convulsions, and electrical seizures. The induction of epilepsy may be related to generation of free radicals, lipid peroxidation of neuronal membranes, increased intracellular calcium concentrations through reverse action of sodium-calcium exchanger/reduced activity of plasma membrane or endoplasmic reticulum calcium ATPases, increased release of excitatory neurotransmitters, including aspartate and glutamate, and increased influx of ions through glutamate receptors. Some of the above effects of iron can be abrogated by inhibitors of phospholipase A₂ (PLA₂) indicating that the damaging effects of iron may be due to perturbation of the lipid environment essential to normal functioning of membrane proteins. Iron in hemoglobin, or by itself, is also likely to be the cause of human epilepsy, in instances where there is increased iron load in the brain. These include subarachnoid hemorrhage, intraparenchymal hemorrhages due to head injury and stroke, malaria, human immunodeficiency virus encephalitis, and possibly, neuroleptic drug use. A reduced level of haptoglobin, a hemoglobin-binding protein, has also been observed in select kindred relatives affected with familial idiopathic epilepsy. An accumulation of iron has been observed in the motor cortex with age, and it is possible that this might contribute to the increased incidence of epilepsy among the elderly. Iron accumulates with time in rat hippocampus after kainate-induced epilepsy. The accumulation occurs in oligodendrocytes, and is likely to be a reflection of the high levels of iron in the extracellular space. The accumulation of iron is correlated with increased expression of the divalent metal transporter-1 in astrocytes in the glial scar and increased expression of heme oxygenase-1 in reactive astrocytes and microglia, as well as degenerating neurons at the edge of the scar. The increased divalent metal transporter-1 expression could lead to increased uptake of iron, followed by redistribution to the extracellular space. In this model, iron is the consequence of epilepsy, although it is possible that it can also be a cause of epilepsy. Further work is necessary to elucidate the effects of lipid peroxidation of the cellular membranes on function of membrane proteins and the role of phospholipases, including PLA₂, in perturbing the lipid environment. The possible presence of iron in the human brain after epilepsy also needs to be elucidated. The causes of dysregulation of iron in the glial scar after neuronal injury need to be studied. In addition, possible beneficial effects of iron chelators, antioxidants that cross the blood-brain barrier, or neuroprotective gene induction on epilepsy, need to be evaluated.

The most labile and most sensitive link in the chain of events that constitute the memory process is the phase of consolidation that follows after acquisition, by which memories are transformed, at a loss, from an unstable into a stable state. Therefore the pharmacology of consolidation has been better studied than that of the other phases of memory (acquisition, storage, retrieval). Pharmacological studies unveiled much information concerning the actual mechanism of consolidation. This involves excitatory glutamatergic and cholinergic muscarinic synapses in the amygdala, medial septum and hippocampus, inhibited by benziodiazepine-regulated GABA-A synapses and modulated by B-noradrenergic terminals. Other additional neutrotransmitter systems, possibly different in each structure, may also intervene. Peripheral hormones reflexly modulate these mechanisms. The amygdala, medial septum and hippocampus process different aspects or components of memories (spatial, aversive, etc.). The entorhinal cortex, which receives projections from these three structures, has a post-consolidational, presumably integrative function. The glutamatergic synapses involved in memory consolidation in the amygdala and hippocampus sustain this late role of the entorhinal cortex through the generation of relatively brief long-term potentiations.