Natural product Kaji-ichigoside F1 exhibits rapid antidepression via activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway.

BACKGROUND: Depression is a common and recurrent neuropsychiatric disorder. Recent studies have shown that the N-methyl-d-aspartate (NMDA) receptor (NMDAR) is involved in the pathophysiology of depression. Previous studies have found that Kaji-ichigoside F1 (KF1) has a protective effect against NMDA-induced neurotoxicity. However, the antidepressant mechanism of KF1 has not been confirmed yet. PURPOSE: In the present study, we aimed to evaluate the rapid antidepressant activity of KF1 and explore the underlying mechanism. STUDY DESIGN: First, we explored the effect of KF1 on NMDA-induced hippocampal neurons and the underlying mechanism. Second, depression was induced in C57BL/6 mice via chronic unpredictable mild stress (CUMS), and the immediate and persistent depression-like behavior was evaluated using the forced swimming test (FST) after a single administration of KF1. Third, the contributions of NMDA signaling to the antidepressant effect of KF1 were investigated using pharmacological interventions. Fourth, CUMS mice were treated with KF1 for 21 days, and then their depression-like behaviors and the underlying mechanism were further explored. METHODS: The FST was used to evaluate immediate and persistent depression-like behavior after a single administration of KF1 with or without NMDA pretreatment. The effect of KF1 on depressive-like behavior was investigated in CUMS mice by treating them with KF1 once daily for 21 days through the sucrose preference test, FST, open field test, and tail suspension test. Then, the effects of KF1 on the morphology and molecular and functional phenotypes of primary neuronal cells and hippocampus of mice were investigated by hematoxylin-eosin staining, Nissl staining, propidium iodide staining, TUNEL staining, Ca2+ imaging, JC-1 staining, ELISA, immunofluorescence analysis, RT-PCR, and Western blot. RESULTS: KF1 could effectively improve cellular viability, reduce apoptosis, inhibit the release of LDH and Ca2+, and increase the mitochondrial membrane potential and the number of dendritic spines numbers in hippocampal neurons. Moreover, behavioral tests showed that KF1 exerted acute and sustained antidepressant-like effects by reducing Glu-levels and ameliorating neuronal damage in the hippocampus. Additionally, in vivo and in vitro experiments revealed that PSD95, Syn1, α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and brain-derived neurotrophic factor (BDNF) were upregulated at the protein level, and BDNF and AMPA were upregulated at the mRNA level. NR1 and NR2A showed the opposite trend. CONCLUSION: These results confirm that KF1 exerts rapid antidepressant effects mainly by activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway. This study serves as a new reference for discovering rapid antidepressants.

Feeding the Brain: Effect of Nutrients on Cognition, Synaptic Function, and AMPA Receptors.

In recent decades, traditional eating habits have been replaced by a more globalized diet, rich in saturated fatty acids and simple sugars. Extensive evidence shows that these dietary factors contribute to cognitive health impairment as well as increase the incidence of metabolic diseases such as obesity and diabetes. However, how these nutrients modulate synaptic function and neuroplasticity is poorly understood. We review the Western, ketogenic, and paleolithic diets for their effects on cognition and correlations with synaptic changes, focusing mainly (but not exclusively) on animal model studies aimed at tracing molecular alterations that may contribute to impaired human cognition. We observe that memory and learning deficits mediated by high-fat/high-sugar diets, even over short exposure times, are associated with reduced arborization, widened synaptic cleft, narrowed post-synaptic zone, and decreased activity-dependent synaptic plasticity in the hippocampus, and also observe that these alterations correlate with deregulation of the AMPA-type glutamate ionotropic receptors (AMPARs) that are crucial to neuroplasticity. Furthermore, we explored which diet-mediated mechanisms modulate synaptic AMPARs and whether certain supplements or nutritional interventions could reverse deleterious effects, contributing to improved learning and memory in older people and patients with Alzheimer's disease.

Calcineurin Controls Hypothalamic NMDA Receptor Activity and Sympathetic Outflow.

RATIONALE: Hypertension is a common and serious adverse effect of calcineurin inhibitors, including cyclosporine and tacrolimus (FK506). Although increased sympathetic nerve discharges are associated with calcineurin inhibitor-induced hypertension, the sources of excess sympathetic outflow and underlying mechanisms remain elusive. Calcineurin (protein phosphatase-2B) is broadly expressed in the brain, including the paraventricular nuclear (PVN) of the hypothalamus, which is critically involved in regulating sympathetic vasomotor tone. OBJECTIVE: We determined whether prolonged treatment with the calcineurin inhibitor causes elevated sympathetic output and persistent hypertension by potentiating synaptic N-methyl-D-aspartate (NMDA) receptor activity in the PVN. METHODS AND RESULTS: Telemetry recordings showed that systemic administration of FK506 (3 mg/kg per day) for 14 days caused a gradual and profound increase in arterial blood pressure in rats, which lasted at least 7 days after discontinuing FK506 treatment. Correspondingly, systemic treatment with FK506 markedly reduced calcineurin activity in the PVN and circumventricular organs, but not rostral ventrolateral medulla, and increased the phosphorylation level and synaptic trafficking of NMDA receptors in the PVN. Immunocytochemistry labeling showed that calcineurin was expressed in presympathetic neurons in the PVN. Whole-cell patch-clamp recordings in brain slices revealed that treatment with FK506 increased baseline firing activity of PVN presympathetic neurons; this increase was blocked by the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Also, treatment with FK506 markedly increased presynaptic and postsynaptic NMDA receptor activity of PVN presympathetic neurons. Furthermore, microinjection of the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist into the PVN of anesthetized rats preferentially attenuated renal sympathetic nerve discharges and blood pressure elevated by FK506 treatment. In addition, systemic administration of memantine, a clinically used NMDA receptor antagonist, effectively attenuated FK506 treatment-induced hypertension in conscious rats. CONCLUSIONS: Our findings reveal that normal calcineurin activity in the PVN constitutively restricts sympathetic vasomotor tone via suppressing NMDA receptor activity, which may be targeted for treating calcineurin inhibitor-induced hypertension.

Conveyance of cortical pacing for parkinsonian tremor-like hyperkinetic behavior by subthalamic dysrhythmia.

Parkinson's disease is characterized by both hypokinetic and hyperkinetic symptoms. While increased subthalamic burst discharges have a direct causal relationship with the hypokinetic manifestations (e.g., rigidity and bradykinesia), the origin of the hyperkinetic symptoms (e.g., resting tremor and propulsive gait) has remained obscure. Neuronal burst discharges are presumed to be autonomous or less responsive to synaptic input, thereby interrupting the information flow. We, however, demonstrate that subthalamic burst discharges are dependent on cortical glutamatergic synaptic input, which is enhanced by A-type K+ channel inhibition. Excessive top-down-triggered subthalamic burst discharges then drive highly correlative activities bottom-up in the motor cortices and skeletal muscles. This leads to hyperkinetic behaviors such as tremors, which are effectively ameliorated by inhibition of cortico-subthalamic AMPAergic synaptic transmission. We conclude that subthalamic burst discharges play an imperative role in cortico-subcortical information relay, and they critically contribute to the pathogenesis of both hypokinetic and hyperkinetic parkinsonian symptoms.

Pathway-Specific Remodeling of Thalamostriatal Synapses in Parkinsonian Mice.

Movement suppression in Parkinson's disease (PD) is thought to arise from increased efficacy of the indirect pathway basal ganglia circuit, relative to the direct pathway. However, the underlying pathophysiological mechanisms remain elusive. To examine whether changes in the strength of synaptic inputs to these circuits contribute to this imbalance, we obtained paired whole-cell recordings from striatal direct- and indirect-pathway medium spiny neurons (dMSNs and iMSNs) and optically stimulated inputs from sensorimotor cortex or intralaminar thalamus in brain slices from control and dopamine-depleted mice. We found that dopamine depletion selectively decreased synaptic strength at thalamic inputs to dMSNs, suggesting that thalamus drives asymmetric activation of basal ganglia circuitry underlying parkinsonian motor impairments. Consistent with this hypothesis, in vivo chemogenetic and optogenetic inhibition of thalamostriatal terminals reversed motor deficits in dopamine-depleted mice. These results implicate thalamostriatal projections in the pathophysiology of PD and support interventions targeting thalamus as a potential therapeutic strategy.

Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors.

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates.

Thalamocortical (TC) connectivity is reorganized by thalamic inputs during postnatal development; however, the dynamic characteristics of TC reorganization and the underlying mechanisms remain unexplored. We addressed this question using dendritic refinement of layer 4 (L4) stellate neurons in mouse barrel cortex (barrel cells) as a model; dendritic refinement of L4 neurons is a critical component of TC reorganization through which postsynaptic L4 neurons acquire their dendritic orientation toward presynaptic TC axon termini. Simultaneous labeling of TC axons and individual barrel cell dendrites allowed in vivo time-lapse imaging of dendritic refinement in the neonatal cortex. The barrel cells reinforced the dendritic orientation toward TC axons by dynamically moving their branches. In N-methyl-D-aspartate receptor (NMDAR)-deficient barrel cells, this dendritic motility was enhanced, and the orientation bias was not reinforced. Our data suggest that L4 neurons have "fluctuating" dendrites during TC reorganization and that NMDARs cell autonomously regulate these dynamics to establish fine-tuned circuits.

Activity-dependent adenosine release may be linked to activation of Na(+)-K(+) ATPase: an in vitro rat study.

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na(+) influxes associated with neuronal signalling activate the Na(+)-K(+) ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na(+). We have simultaneously imaged intracellular Na(+) and measured adenosine release. The accumulation of intracellular Na(+) during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca(2+), and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na(+)-K(+) ATPase in the release of adenosine. Under conditions which caused a Na(+) influx, brief applications of ouabain increased the accumulation of intracellular Na(+) but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na(+)-K(+) ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na(+)-K(+) ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep.

Inhibitory effects of acorn extract on glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Various effects of acorn extract have been reported including antioxidant activity, cytotoxicity against cancer cells, and the levels of acetylcholine and its related enzyme activities in the dementia mouse models. However, it is unclear whether acorn extract inhibits glutamate-induced calcium signaling in hippocampal neurons. This study was an investigation into the effect of acorn extract on intracellular free Ca concentrations ([Ca]) in cultured rat hippocampal neurons using fura-2-based digital calcium imaging and photometry. Hippocampal neurons were used between 10 and 14 d in culture from embryonic day-18 rats. Treatment with acorn extract (1 µg/mL to 1 mg/mL) for 30 min inhibited glutamate (100 µM)-induced [Ca] increases in a dose-dependent manner (IC=46.9 µg/mL). After depletion of intracellular Ca stores by treatment with the inhibitor endoplasmic reticulum Ca-ATPase, thapsigargin (1 µM), treatment with acorn extract (50 µg/mL) for 30 min decreased the subsequent glutamate-induced [Ca] increases. Acorn extract significantly inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (30 µM)-induced [Ca] increases. In addition, acorn extract inhibited the AMPA-induced [Ca] responses in the presence of 1 µM nimodipine. Acorn extract also significantly inhibited N-methyl-D-aspartate (100 µM)-induced [Ca] increases. Acorn extract significantly inhibited 50 mM KCl -induced [Ca] increases. Acorn extract significantly inhibited (S)-3,5-dihydroxyphenylglycine-induced [Ca] responses. Moreover, acorn extract almost completely blocked synaptically mediated [Ca] spikes induced by decreasing extracellular Mg concentration to 0.1 mM. These results suggest that acorn extract inhibits synaptically induced frequent [Ca] spikes through multiple pathways such as ionotropic glutamate receptors, voltage-gated Ca channels and metabotropic glutamate receptors in cultured rat hippocampal neurons.

Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 µm) and distal dendrites (∼130 µm dendrite length), but intervening midlengths of dendrite (∼27-130 µm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.

In vitro studies of the influence of glutamatergic agonists on the Na+,K(+)-ATPase and K(+)-p-nitrophenylphosphatase activities in the hippocampus and frontal cortex of rats.

BACKGROUND: The overstimulation of excitatory glutamatergic neurotransmission and the inhibition of Na(+),K(+)-ATPase enzymatic activity have both been implicated in neurotoxicity and are possibly related to the pathogenesis of epilepsy and neurodegenerative disorders. In the present study, we investigated whether glutamatergic stimulation by the glutamatergic agonists glutamate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) modulates the Na(+),K(+)-ATPase and the K(+)-p-nitrophenylphosphatase activities in the crude synaptosomal fraction of the hippocampus and the frontal cortex of rats. RESULTS: Our results demonstrated that these glutamatergic agonists did not influence the activities of Na(+),K(+)-ATPase or K(+)-p-nitrophenylphosphatase in the brain structures analyzed. Assays with lower concentrations of ATP to analyze the preferential activity of the Na(+),K(+)-ATPase isoform with high affinity for ATP did not show any influence either. CONCLUSIONS: These findings suggest that under our experimental conditions, the stimulation of glutamatergic receptors does not influence the kinetics of the Na(+),K(+)-ATPase enzyme in the hippocampus and frontal cortex.

Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse.

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

An integrative view of mechanisms underlying generalized spike-and-wave epileptic seizures and its implication on optimal therapeutic treatments.

Many types of epileptic seizures are characterized by generalized spike-and-wave discharges. In the past, notable effort has been devoted to understanding seizure dynamics and various hypotheses have been proposed to explain the underlying mechanisms. In this paper, by taking an integrative view of the underlying mechanisms, we demonstrate that epileptic seizures can be generated by many different combinations of synaptic strengths and intrinsic membrane properties. This integrative view has important medical implications: the specific state of a patient characterized by a set of biophysical characteristics ultimately determines the optimal therapeutic treatment. Through the same view, we further demonstrate the potentiation effect of rational polypharmacy in the treatment of epilepsy and provide a new angle to resolve the debate on polypharmacy. Our results underscore the need for personalized medicine and demonstrate that computer modeling and simulation may play an important role in assisting the clinicians in selecting the optimal treatment on an individual basis.

Dopamine D1 receptor blockage potentiates AMPA-stimulated luteinising hormone release in the goldfish.

Previous microarray analyses of the goldfish hypothalamus led us to hypothesise that dopamine could potentially inhibit the excitatory effects of glutamate on luteinising hormone (LH). Post-spawning female goldfish were pre-treated (-4.5 h) with either saline (C; control), SCH 23390 (S; D(1) -receptor antagonist) or sulpiride (L; D(2) -receptor antagonist), followed by an i.p. injection, at -0.5 h, of saline or the glutamate agonist AMPA (A, SA or LA). Blood, hypothalamus and telencephalon tissues were collected. Serum LH was not affected in the S, L, A, or LA groups relative to control as determined by radioimmunoassay. The SA group, however, showed a 289% (P<0.0005) increase in serum LH compared to either treatment alone or control. Real-time reverse transcriptase-polymerase chain reaction identified the mRNAs for ionotropic (Gria2a, Gria4) glutamate receptor subunits, activin ßa, isotocin, and cGnRH-II as being significantly affected by some of the treatments. The same experiment conducted with sexually-regressed female fish showed a very different LH profile, indicating that this mechanism is seasonally-dependent. We also show that i.p. injection of 1 µg/g isotocin was able to increase LH levels by 167% in sexually regressed female fish relative to controls. Taken together, these results demonstrate that blockage of the D(1) receptor primes post-spawning goldfish for AMPA-stimulated LH release, and provides further insights into the central regulation of reproduction.

Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn.

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca(2+) permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca(2+) imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca(2+)-impermeable receptors. Maintenance of complete Freund's adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca(2+)](i) transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca(2+) permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca(2+)-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca(2+)-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.

Emergence of cortical inhibition by coordinated sensory-driven plasticity at distinct synaptic loci.

Feedforward GABAergic inhibition sets the dendritic integration window, thereby controlling timing and output in cortical circuits. However, the manner in which feedforward inhibitory circuits emerge is unclear, despite this being a critical step for neocortical development and function. We found that sensory experience drove plasticity of the feedforward inhibitory circuit in mouse layer 4 somatosensory barrel cortex in the second postnatal week via two distinct mechanisms. First, sensory experience selectively strengthened thalamocortical-to-feedforward interneuron inputs via a presynaptic mechanism but did not regulate other inhibitory circuit components. Second, experience drove a postsynaptic mechanism in which a downregulation of a prominent thalamocortical NMDA excitatory postsynaptic potential in stellate cells regulated the final expression of functional feedforward inhibitory input. Thus, experience is required for specific, coordinated changes at thalamocortical synapses onto both inhibitory and excitatory neurons, producing a circuit plasticity that results in maturation of functional feedforward inhibition in layer 4.

High-throughput screening in embryonic stem cell-derived neurons identifies potentiators of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type glutamate receptors.

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 x 10(6) compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10(6) compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.

Self-sustained asynchronous irregular states and Up-Down states in thalamic, cortical and thalamocortical networks of nonlinear integrate-and-fire neurons.

Randomly-connected networks of integrate-and-fire (IF) neurons are known to display asynchronous irregular (AI) activity states, which resemble the discharge activity recorded in the cerebral cortex of awake animals. However, it is not clear whether such activity states are specific to simple IF models, or if they also exist in networks where neurons are endowed with complex intrinsic properties similar to electrophysiological measurements. Here, we investigate the occurrence of AI states in networks of nonlinear IF neurons, such as the adaptive exponential IF (Brette-Gerstner-Izhikevich) model. This model can display intrinsic properties such as low-threshold spike (LTS), regular spiking (RS) or fast-spiking (FS). We successively investigate the oscillatory and AI dynamics of thalamic, cortical and thalamocortical networks using such models. AI states can be found in each case, sometimes with surprisingly small network size of the order of a few tens of neurons. We show that the presence of LTS neurons in cortex or in thalamus, explains the robust emergence of AI states for relatively small network sizes. Finally, we investigate the role of spike-frequency adaptation (SFA). In cortical networks with strong SFA in RS cells, the AI state is transient, but when SFA is reduced, AI states can be self-sustained for long times. In thalamocortical networks, AI states are found when the cortex is itself in an AI state, but with strong SFA, the thalamocortical network displays Up and Down state transitions, similar to intracellular recordings during slow-wave sleep or anesthesia. Self-sustained Up and Down states could also be generated by two-layer cortical networks with LTS cells. These models suggest that intrinsic properties such as adaptation and low-threshold bursting activity are crucial for the genesis and control of AI states in thalamocortical networks.

Extracts of Liriopsis tuber protect AMPA induced brain damage and improve memory with the activation of insulin receptor and ERK I/II.

The brain insulin receptor and ERK I/II are known to play an important role in memory formation and neuroprotection. A series of experiments was designed to explore if Liriopsis tuber (LT) extracts could exhibit neuroprotection and memory enhancing actions. LT was extracted with 70% methanol and subsequently fractionated into chloroform (fraction C), chloroform/methanol-(3:1) (fraction CM), methanol-soluble (fraction M) and methanol-insoluble, water-soluble fractions (fraction A). The LT fractions (T, C, M, A) significantly inhibited the cortical depolarization induced by AMPA in cortical slices of rats. In addition, these fractions were also effective in promoting memory in the passive avoidance test in mice. To gain insight into the mechanism of memory enhancing effects by Liriopsis tuber extracts, the activities of hippocampal insulin receptors and ERK I/II were tested in rats. Extract of LT (T) dramatically stimulated tyrosine phosphorylation of the insulin receptor, while fraction C of LT also significantly stimulated the same. In addition, ERK I/II were stimulated and cholinesterase activities were inhibited by fractions T, C, M and A in the rat hippocampus. These results suggest that Liriopsis tuber extracts may exert neuroprotection and memory enhancing effects via activation of the insulin receptor and ERK I/II as well as inhibiting cholinesterase.

Antipsychotic drugs reverse the AMPA receptor-stimulated release of 5-HT in the medial prefrontal cortex.

The prefrontal cortex (PFC) is involved in the pathophysiology of schizophrenia. PFC neuronal activity is modulated by monoaminergic receptors for which antipsychotic drugs display moderate-high affinity, such as 5-HT(2A) and alpha(1)-adrenoceptors. Conversely, PFC pyramidal neurons project to and modulate the activity of raphe serotonergic neurons and serotonin (5-HT) release. Under the working hypothesis that atypical antipsychotic drugs may partly exert their action in PFC, we assessed their action on the in vivo 5-HT release evoked by increasing glutamatergic transmission in rat medial PFC (mPFC). This was achieved by applying S-AMPA in mPFC (reverse dialysis) or by disinhibiting thalamic excitatory afferents to mPFC with bicuculline. The application of haloperidol, chlorpromazine, clozapine and olanzapine in mPFC by reverse dialysis (but not reboxetine or diazepam) reversed the S-AMPA-evoked local 5-HT release. Likewise, the local (in mPFC) or systemic administration of these antipsychotic drugs reversed the increased prefrontal 5-HT release produced by thalamic disinhibition. These effects were shared by the 5-HT(2A) receptor antagonist M100907 and the alpha(1)-adrenoceptor antagonist prazosin. However, raclopride (DA D2 antagonist) had very modest effects. These results suggest that, besides their action in limbic striatum, antipsychotic drugs may attenuate glutamatergic transmission in PFC, possibly by interacting with 5-HT(2A) and/or alpha(1)-adrenoceptors.