Activation Of AMPA Receptors Research Articles

Strictly regulated agonist-dependent activation of AMPA-R is the key characteristic of TAK-653 for robust synaptic responses and cognitive improvement

Agonistic profiles of AMPA receptor (AMPA-R) potentiators may be associated with seizure risk and bell-shaped dose-response effects. Here, we report the pharmacological characteristics of a novel AMPA-R potentiator, TAK-653, which exhibits minimal agonistic properties. TAK-653 bound to the ligand binding domain of recombinant AMPA-R in a glutamate-dependent manner. TAK-653 strictly potentiated a glutamate-induced Ca2+ influx in hGluA1i-expressing CHO cells through structural interference at Ser743 in GluA1. In primary neurons, TAK-653 augmented AMPA-induced Ca2+ influx and AMPA-elicited currents via physiological AMPA-R with little agonistic effects. Interestingly, TAK-653 enhanced electrically evoked AMPA-R-mediated EPSPs more potently than AMPA (agonist) or LY451646 (AMPA-R potentiator with a prominent agonistic effect) in brain slices. Moreover, TAK-653 improved cognition for both working memory and recognition memory, while LY451646 did so only for recognition memory, and AMPA did not improve either. These data suggest that the facilitation of phasic AMPA-R activation by physiologically-released glutamate is the key to enhancing synaptic and cognitive functions, and nonselective activation of resting AMPA-Rs may negatively affect this process. Importantly, TAK-653 had a wide safety margin against convulsion; TAK-653 showed a 419-fold (plasma Cmax) and 1017-fold (AUC plasma) margin in rats. These findings provide insight into a therapeutically important aspect of AMPA-R potentiation.

Biophysical and synaptic properties of NMDA receptors in the lateral habenula

Excitatory synaptic transmission in the lateral habenula (LHb), an evolutionarily ancient subcortical structure, encodes aversive stimuli and affective states. Habenular glutamatergic synapses contribute to these processes partly through the activation of AMPA receptors. Yet, N-methyl-d-aspartate receptors (NMDARs) are also expressed in the LHb and support the emergence of depressive symptoms. Indeed, local NMDAR blockade in the LHb rescues anhedonia and behavioral despair in rodent models of depression. However, the subunit composition and biophysical properties of habenular NMDARs remain unknown, thereby hindering their study in the context of mental health. Here, we performed electrophysiological recordings and optogenetic-assisted circuit mapping in mice, to study pharmacologically-isolated NMDAR currents in LHb neurons that receive innervation from different brain regions (entopeduncular nucleus, lateral hypothalamic area, bed nucleus of the stria terminalis, or ventral tegmental area). This systematic approach revealed that habenular NMDAR currents are sensitive to TCN and ifenprodil – drugs that specifically inhibit GluN2A- and GluN2B-containing NMDARs, respectively. Whilst these pharmacological effects were consistently observed across inputs, we detected region-specific differences in the current-voltage relationship and decay time of NMDAR currents. Finally, inspired by the firing of LHb neurons in vivo, we designed a burst protocol capable of eliciting calcium-dependent long-term potentiation of habenular NMDAR transmission ex vivo. Altogether, we define basic biophysical and synaptic properties of NMDARs in LHb neurons, opening new avenues for studying their plasticity processes in physiological as well as pathological contexts.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

HDACi protects against vascular cognitive impairment from CCH injury via induction of BDNF-related AMPA receptor activation.

We previously showed a hydroxamic acid‐based histone deacetylase inhibitor (HDACi), compound 13, provides neuroprotection against chronic cerebral hypoperfusion (CCH) both in vitro under oxygen‐glucose deprivation (OGD) conditions and in vivo under bilateral common carotid artery occlusion (BCCAO) conditions. Intriguingly, the protective effect of this HDACi is via H3K14 or H4K5 acetylation–mediated differential BDNF isoform activation. BDNF is involved in cell proliferation and differentiation in development, synaptic plasticity and in learning and memory related with receptors or synaptic proteins. B6 mice underwent BCCAO and were randomized into 4 groups; a sham without BCCAO (sham), BCCAO mice injected with DMSO (DMSO), mice injected with HDACi‐compound 13 (compound 13) and mice injected with suberoylanilide hydroxamic acid (SAHA). The cortex and hippocampus of mice were harvested at 3 months after BCCAO, and levels of BDNF, AMPA receptor and dopamine receptors (D1, D2 and D3) were studied using Western blotting analysis or immunohistochemistry. We found that the AMPA receptor plays a key role in the molecular mechanism of this process by modulating HDAC. This protective effect of HDACi may be through BDNF; therefore, activation of this downstream signalling molecule, for example by AMPA receptors, could be a therapeutic target or intervention applied under CCH conditions.

Co-administration of cannabidiol and ketamine induces antidepressant-like effects devoid of hyperlocomotor side-effects

Background and PurposeAlthough useful as a rapid-acting antidepressant drug, ketamine is known to induce psychotomimetic effects, which may interfere with its therapeutic use. Cannabidiol (CBD) is a non-psychostimulant compound from Cannabis sativa, which has shown promising antidepressant effects without inducing hyperlocomotion. AMPA receptor activation is involved in the antidepressant effect induced by ketamine, but its relevance for the effects of CBD is not known. Moreover, given that CBD has antipsychotic and antidepressant properties, it is unknown whether adding CBD to ketamine could potentiate the antidepressant properties of ketamine while also attenuating its psychostimulant effects. Experimental approachS-Ketamine (2.5, 3, 5, 10, 30 mg/kg) and cannabidiol (3, 10, 30 mg/kg) were administered alone or in combination to male Swiss mice. Independent groups received NBQX (AMPA receptor antagonist) 5 min before administration of CBD or S-ketamine. The antidepressant-like effect was assessed in the forced swimming test (FST), and the open field test (OFT) evaluated the psychostimulant effect. Key resultsCBD induced significant dose-dependent antidepressant effects without causing hyperlocomotion in the OFT. S-ketamine produced an antidepressant effect associated with hyperlocomotion in the higher dose. NBQX inhibited the antidepressant effect of both ketamine and CBD. Pretreatment with CBD (10 mg/kg) attenuated the ketamine-induced hyperlocomotion while preserving its antidepressant effect. ConclusionAND IMPLICATIONS: Similar to ketamine, the antidepressant-like effect elicited by CBD involves AMPA receptor activation. Additionally, CBD prevents the hyperlocomotion induced by S-ketamine without affecting its antidepressant-like effect. Our findings suggest that CBD and ketamine's combined administration can be a promising therapeutic strategy for achieving an appropriate antidepressant effect without unwanted side-effects.This article is part of the special issue on ‘Cannabinoids'.

Calcium-permeable AMPA receptor activity and GluA1 trafficking in the basolateral amygdala regulate operant alcohol self-administration.

Addiction is viewed as maladaptive glutamate-mediated neuroplasticity that is regulated, in part, by calcium-permeable AMPA receptor (CP-AMPAR) activity. However, the contribution of CP-AMPARs to alcohol-seeking behavior remains to be elucidated. We evaluated CP-AMPAR activity in the basolateral amygdala (BLA) as a potential target of alcohol that also regulates alcohol self-administration in C57BL/6J mice. Operant self-administration of sweetened alcohol increased spontaneous EPSC frequency in BLA neurons that project to the nucleus accumbens as compared with behavior-matched sucrose controls indicating an alcohol-specific upregulation of synaptic activity. Bath application of the CP-AMPAR antagonist NASPM decreased evoked EPSC amplitude only in alcohol self-administering mice indicating alcohol-induced synaptic insertion of CP-AMPARs in BLA projection neurons. Moreover, NASPM infusion in the BLA dose-dependently decreased the rate of operant alcohol self-administration providing direct evidence for CP-AMPAR regulation of alcohol reinforcement. As most CP-AMPARs are GluA1-containing, we asked if alcohol alters the activation state of GluA1-containing AMPARs. Immunocytochemistry results showed elevated GluA1-S831 phosphorylation in the BLA of alcohol as compared with sucrose mice. To investigate mechanistic regulation of alcohol self-administration by GluA1-containing AMPARs, we evaluated the necessity of GluA1 trafficking using a TET-ON AAV encoding a dominant-negative GluA1 c-terminus (GluA1ct) that blocks activity-dependent synaptic delivery of native GluA1-containing AMPARs. GluA1ct expression in the BLA reduced alcohol self-administration with no effect on sucrose controls. These results show that CP-AMPAR activity and GluA1 trafficking in the BLA mechanistically regulate the reinforcing effects of sweetened alcohol. Pharmacotherapeutic targeting these mechanisms of maladaptive neuroplasticity may aid medical management of alcohol use disorder.

Enhancing Synaptic Plasticity in vitro using Novel Ketamine Derivatives

Loss of synaptic plasticity in the brain has been linked to aging-associated cognitive decline and neurodegeneration, thus providing a promising target for therapeutic interventions. Ketamine, a drug applied for analgesia in emergency medicine and in treatment-resistant depression, and some of its metabolites rapidly induce synaptic plasticity. However, ketamine cannot be reasonably applied as a neuroprotective agent given its side-effect profile. With the aim to dissociate pro-neuroplastic actions from NMDA receptor-mediated psychotropic side effects we studied novel ketamine derivatives in vitro. We applied ketamine and different ketamine derivatives at various concentrations and incubation durations to primary hippocampal neurons cultivated until maturity of synaptic development. Vesicle-associated membrane protein 2 (VAMP-2) immunostaining served as a measure for pre-synaptic density. For treatment with several candidates, e.g. compound HW-273, pre-synapse number was enhanced in a concentration-dependent manner (Figure 1). Furthermore, enhanced intensity measures of post-synapse immunostaining for GluA2 (AMPA receptor subunit) in compound-treated cells indicated elevated AMPA receptor activation at low doses (0.5-1 µM), whereas for ketamine a higher dose was required for comparable effects (Figure 2). In addition, treatment with high compound concentrations showed no obvious cytotoxic effects, opposite to treatment with ketamine. We conclude that novel ketamine derivatives potently augment neuronal plasticity in a concentration-dependent fashion. Our study provides preliminary evidence that the potentially beneficial effect on neuronal plasticity can be dissociated from NMDA receptor engagement, which could pave the way for development of new neuroprotective therapies with less unwanted side-effects.

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling.

Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway).

P2X4 receptor in the dorsal horn contributes to BDNF/TrkB and AMPA receptor activation in the pathogenesis of remifentanil-induced postoperative hyperalgesia in rats

The mechanism underlying the high incidence of remifentanil-induced postoperative hyperalgesia is unclear. Also, no effective prevention method exists. Inflammatory pain–related studies showed that P2X4 purinergic receptors (P2X4Rs) in the dorsal horn of the spinal cord and dorsal root ganglia are essential for maintaining allodynia caused by inflammation. However, little is known about its role in opioid-induced hyperalgesia. This study aimed to determine the role of P2X4R and related signaling pathways in the remifentanil-induced postoperative hyperalgesia (RIH) model. The study simulated the remifentanil infusion and surgical incision during general anesthesia. The mRNA and protein expression level of P2X4R in rats with RIH model increased from 2 h to 48 h after the surgery. The administration of P2X4R inhibitors prevented the occurrence of RIH, resulting in a reduction in mechanical and thermal pain. Moreover, P2X4R was involved in RIH in male and female rats, indicating no sex-specific difference. P2X4R also increased the expression of AMPA receptor subunit GluA1 in a brain-derived neurotrophic factor (BDNF) / tyrosine receptor kinase B (TrkB) dependent manner. The results from whole-cell patch-clamp recording suggested that P2X4R also regulated AMPA receptor-mediated miniature excitatory postsynaptic currents and participated in the synaptic plasticity of spinal dorsal horn neurons. In summary, P2X4R was involved in AMPAR expression, electrophysiological function, and synaptic plasticity of spinal dorsal horn neurons through BDNF/TrkB signaling. This might be the mechanism underlying RIH, and hence inhibition of P2X4R might be a potential treatment strategy.

Age-related vulnerability to nigral dopaminergic degeneration in rats via Zn2+-permeable GluR2-lacking AMPA receptor activation

On the basis of the evidence that extracellular Zn2+ influx induced with AMPA causes Parkinson's syndrome in rats that apomorphine-induced movement disorder emerges, here we used a low dose of AMPA, which does not increase intracellular Zn2+ level in the substantia nigra pars compacta (SNpc) of young adult rats, and tested whether intracellular Zn2+ dysregulation induced with AMPA is accelerated in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome. When AMPA (1 mM) was injected at the rate of 0.05 μl/min for 20 min into the SNpc, intracellular Zn2+ level was increased in the SNpc of aged rats followed by increase in turning behavior in response to apomorphine and nigral dopaminergic degeneration. In contrast, young adult rats do not show movement disorder and nigral dopaminergic degeneration, in addition to no increase in intracellular Zn2+. In aged rats, movement disorder and nigral dopaminergic degeneration were rescued by co-injection of either extracellular (CaEDTA) or intracellular (ZnAF-2DA) Zn2+ chelators. 1-Naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors blocked increase in intracellular Zn2+ in the SNpc of aged rats followed by rescuing nigral dopaminergic degeneration. The present study indicates that intracellular Zn2+ dysregulation is accelerated by Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptor activation in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome.

Technologic Considerations of Creation Dosage Form of Original Substance 3,7-diazabicyclo[3.3.1]nonane

Introduction. An innovative pharmaceutical substance based on the 3,7-diazabicyclo[3.3.1]nonane derivative provides long-term activation of AMPA receptors and the production of neurotrophic factors, which makes it possible to use it for the treatment of cognitive impairments and rehabilitation of patients who have undergone acute brain hypoxia. Given that this substance is able to be absorbed through the walls of the gastrointestinal tract and pass through the blood-brain barrier, there are no restrictions on the development of an oral dosage form. In addition, the oral dosage form has significant advantages when used in geriatric and pediatric practice.Aim. The aim of the work was to carry out a comparative study of the possibility of using gelatin and hypromellose capsules for the development of the composition and technology for obtaining a dosage form containing a pharmaceutical substance based on a 3,7-diazabicyclo[3.3.1]nonane derivative.Materials and methods. The study of the «Dissolution» test of gelatinous and hypromellose capsules was carried out on an ERWEKA DT 720 «Paddle stirrer» apparatus at a stirrer rotation speed of 50 rpm in three media: pH 1.2, pH 4.5 and pH 6.8. The content of the substance in each sample was determined by high performance liquid chromatography with UV detection.Results and discussion. The results of the development and testing of capsules containing an original pharmaceutical substance of nootropic action based on a derivative of 3,7-diazabicyclo[3.3.1]nonane, which has low pharmaceutical and technological characteristics and is practically insoluble in water, are presented. The D/S value was ≥250.00 ml in each physiological pH range. The results of determining the dissolution of the developed dosage form in three media at pH values of 1.2, 4.5 and 6.8 showed a positive effect of the technology used on the solubility of the substance.Conclusion. A significant increase in the solubility of the practically insoluble substance of the 3,7-diazabicyclo[3.3.1]nonane derivative in the developed dosage form was shown, which is the result of the use of a well-founded complex of excipients and the technology of moisture-activated granulation. According to the results obtained, (77.60 ± 2.50) % of the substance passes into the dissolution medium with a pH of 4.5 in 45 minutes. The research results are used to develop a technological scheme for obtaining a finished dosage form, its indicators and quality standards.

AMPA receptors mediate the pro-cognitive effects of electrical and optogenetic stimulation of the medial prefrontal cortex in antidepressant non-responsive Wistar–Kyoto rats

Background:The chronic mild stress (CMS) procedure is a widely used animal model of depression, and its application in Wistar–Kyoto (WKY) rats has been validated as a model of antidepressant-refractory depression. While not responding to chronic treatment with antidepressant drugs, WKY rats do respond to acute deep brain stimulation (DBS) of the medial prefrontal cortex (mPFC). In antidepressant-responsive strains there is evidence suggesting a role for AMPA subtype of glutamate receptor in the action mechanism of both antidepressants and DBS.Methods:Animals were subjected to CMS for 6 to 8 weeks; sucrose intake was monitored weekly and novel object recognition (NOR) test was conducted following recovery from CMS. Wistars were treated chronically with venlafaxine (VEN), while WKY were treated acutely with either DBS, optogenetic stimulation (OGS) of virally-transduced (AAV5-hSyn-ChR2-EYFP) mPFC or ventral hippocampus, or acute intra-mPFC injection of the AMPA receptor positive allosteric modulator CX-516. The AMPA receptor antagonist NBQX was administered, at identical sites in mPFC, immediately following the exposure trial in the NOR.Results:Sucrose intake and NOR were suppressed by CMS, and restored by VEN in Wistars and by DBS, OGS, or CX-516 in WKY. However, OGS of the ventral hippocampal afferents to mPFC was ineffective. A low dose of NBQX selectively blocked the procognitive effect of VEN, DBS and OGS.Conclusions:These results suggest that activation of AMPA receptors in the mPFC represents a common pathway for the antidepressant effects of both conventional (VEN) and novel (DBS, OGS) antidepressant modalities, in both antidepressant responsive (Wistar) and antidepressant-resistant (WKY) rats.

Sequential Activation of AMPA Receptors and Glial Cells in a Pain Model of Lumbar Spine Disc Herniation.

ObjectiveTo investigate the glial cell and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor activity after surgery for disc herniation pain model.MethodsIn total, 83 Sprague-Dawley rats were randomly assigned to the following groups: control (n=16), sham-operated (n=4), rats for pain behavior evaluation (n=3), nucleus pulposus-exposed groups for AMPA receptors (n=30), and glial cell (n=30). The rats were tested for mechanical allodynia; immunohistochemical staining for AMPA receptors (GluA1 and GluA2) and glial cells (OX-42 and glial fibrillary acid protein [GFAP]) in the spinal dorsal horn was performed on postoperative days 3, 7, and 14.ResultsMechanical withdrawal thresholds decreased after surgery, and this effect was maintained for up to 14 days. Immunohistochemical expression of GluA1 and GluA2 in the spinal dorsal horn had increased quantitatively on postoperative days 3 and 7 (p<0.05) to levels similar to that of the controls on postoperative day 14. Moreover, immunohistochemical expression of OX-42 and GFAP showed similar changes to AMPA receptors after surgery. Although the activity of AMPA receptors and glial cells achieved normalcy, the mechanical withdrawal threshold of the hind paw remained decreased 38 days after surgery.ConclusionThe rat model of lumbar disc herniation showed increased expression of AMPA receptor and glial cell activity in the spinal dorsal horn 3 and 7 days after surgery, which deceased to control levels at 14 days. The AMPA receptors and glial cell activations showed similar patterns after disc herniation surgery.

Transient Stimulation with Psychoplastogens Is Sufficient to Initiate Neuronal Growth.

Cortical neuron atrophy is a hallmark of depression and includes neurite retraction, dendritic spine loss, and decreased synaptic density. Psychoplastogens, small molecules capable of rapidly promoting cortical neuron growth, have been hypothesized to produce long-lasting positive effects on behavior by rectifying these deleterious structural and functional changes. Here we demonstrate that ketamine and LSD, psychoplastogens from two structurally distinct chemical classes, promote sustained growth of cortical neurons after only short periods of stimulation. Furthermore, we show that psychoplastogen-induced cortical neuron growth can be divided into two distinct epochs: an initial stimulation phase requiring TrkB activation and a growth period involving sustained mTOR and AMPA receptor activation. Our results provide important temporal details concerning the molecular mechanisms by which next-generation antidepressants produce persistent changes in cortical neuron structure, and they suggest that rapidly excreted psychoplastogens might still be effective neurotherapeutics with unique advantages over compounds like ketamine and LSD.

P2X7 receptors exert a permissive effect on the activation of presynaptic AMPA receptors in rat trigeminal caudal nucleus glutamatergic nerve terminals

BackgroundPurine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN).MethodsNerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions.ResultsSynaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this “excess” release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist.ConclusionsP2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

Effect of Neuropeptide Cyclo-L-Prolylglycine on Cell Proliferative Activity.

Endogenous neuropeptide cyclo-L-prolylglycine possesses mnemotropic and neuroprotective properties, which can result from its positive effect on the level of brain-derived neurotrophic factor and modulation of activity of insulin-like growth factor-1 and AMPA receptors. For detection of possible mitogenic action of cyclo-L-prolylglycine, we analyzed its effect on proliferative activity of HEK293 and SH-SY5Y cells assessed by expression of Ki-67 proliferation marker, cell cycle examination, and incorporation of modified nucleotide analog EdU into DNA. Cyclo-L-prolylglycine did not affect the level of Ki-67 in examined cell lines and distribution of the cells over G1 and G2 phases of the cell cycle, although it insignificantly reduced the percentage of S phase cells, which attested to the absence of intrinsic mitogenic activity of the peptide. At the same time, cyclo-L-prolylglycine reduced the number of the early apoptotic cells, which can be a mechanisms of its protective action.

Nature and nurture of sexual partner preference: Teachings from prenatal administration of acetaminophen in male rats

The organizational-activational hypothesis indicates that activation of adult sexual behavior in males depends on organization of the masculine brain during the perinatal sensitive period. In the medial preoptic area such masculinization depends on a neuroendocrine cascade that includes exposure to testosterone, aromatization to estradiol, activation of estrogen receptors, synthesis of cyclooxygenase (COX), increase of prostaglandins, release of glutamate, and activation of AMPA receptors that result in the formation of more dendritic spines. Thus, in the present study we assessed the sexual partner preference (SPP) of adult male rats prenatally treated with acetaminophen (APAP), an analgesic/antipyretic drug that inhibits COX-2 and is commonly used and prescribed during pregnancy. Female rats received either saline (2 ml/kg s.c.) or APAP (50 mg/kg s.c.) every 12 h, during days 16–20 of pregnancy. At postnatal day PD60 half of the male offspring were exposed to sexual experience with receptive females during 5 trials, and the other half remained sexually naïve. At PD90 all them were tested for SPP with one sexually receptive female and one stud male. The results indicated that only APAP-naïve males failed to display SPP. However, APAP-experienced males displayed SPP for females. We discuss the effects of prenatal APAP in the disruption of unconditioned responses towards females (nature mechanisms), and the effects of sexual experience (nurture mechanisms) in the development of conditioned heterosexual preference.

SUN-247 GABA Receptor-Dependent Inhibition and Excitation of GnRH Neuron Dendrons near the Median Eminence in Mice

The gonadotrophin-releasing hormone (GnRH) neuron cell bodies are scattered throughout the basal forebrain but funnel their projections to the median eminence to control fertility in all mammals. In mice, these long projections, termed “dendrons”, have characteristics of both dendrites and axons and are found to receive large numbers of synaptic inputs just prior to entering the median eminence. While the effects of many neurotransmitters have been documented at the GnRH neuron cell body, very little is known about the neural control of the distal dendron.To examine the role of amino acid neurotransmitters in the regulation of the thin GnRH neuron distal dendrons, we used confocal microscopy in combination with real-time GCaMP6 calcium imaging in acute horizontal brain slices. Adult male and female GnRH-Cre mice were given stereotaxic injections of a Cre-dependent AAV expressing GCaMP6s and, > 3 weeks later, the brain removed and a single thick horizontal brain slice prepared that included the base of the brain and median eminence. The intracellular calcium levels of multiple dendrons were monitored simultaneously while puffs of amino acid receptor agonists were applied. Surprisingly, GABA exhibited a dual action on calcium concentrations with an initial transient increase followed by a prolonged decrease and subsequent rebound. The administration of GABAA and GABAB receptor agonist and antagonists revealed that the transient increase resulted from a quickly desensitizing activation of GABAA receptors while the decrease and subsequent rebound was dependent upon the slower kinetics of the GABAB receptor. Kisspeptin exerts a potent long-lasting elevation of calcium levels in GnRH dendrons and this was robustly inhibited by subsequent GABAB receptor activation. In contrast to GABA, glutamate was not found to have any impact on intracellular calcium levels in the distal dendrons and, similarly, the activation of NMDA or AMPA receptors was found to be ineffective.The results show that GABA exerts particularly potent modulatory actions upon basal and evoked calcium concentrations within the GnRH neuron distal dendron. This suggests that acute brief activation of the dendron by GABA would facilitate GnRH release through GABAA receptor activation whereas prolonged GABA release would suppress activity through the GABAB receptor. As such, GABA inputs to the distal dendron appear likely to play a major role in the control of GnRH secretion in the mouse.

Exploring the form- And time-dependent effect of low-frequency electromagnetic fields on maintenance of hippocampal long-term potentiation.

Low-frequency electromagnetic field (LF-EMF) stimulation is an emerging neuromodulation tool that is attracting more attention because of its non-invasive and well-controlled characteristics. However, the effect of different LF-EMF features including the forms and the time of addition on neuronal activity has not been completely understood. In this study, we used multi-electrode array (MEA) systems to develop a flexible in vitro magnetic stimulation device with plug-and-play features that allows for real-time delivery of LF-EMFs to biological tissues. Crucially, the method enables different forms of LF-EMF to be added at any time to a long-term potentiation (LTP) experiment without interrupting the process of LTP induction. We demonstrated that the slope of field excitatory postsynaptic potentials (fEPSPs) decreased significantly under post or priming uninterrupted sine LF-EMFs. The fEPSPs slope would continue to decline significantly when LF-EMFs were added two times with a 20-min interval. Paired-pulse ratio (PPR) was analyzed and the results reflected that LF-EMFs induced LTP was expressed postsynaptically. The results of pharmacological experiments indicated that AMPA receptor activity was involved in the process of LTP loss caused by post-LF-EMFs. Moreover, the effect of priming sine or Quadripulse stimulation (QPS)-patterned LF-EMFs depended on the time interval between the end of LF-EMF and the beginning of baseline recording. Interestingly, the effect of sine LF-EMFs on LTP would not disappear within 120min, while the impact of QPS-patterned LF-EMFs on LTP might disappear after 90min. These results indicated that LF-EMF might have a form- and time-dependent effect on LTP.

Reduction of AMPA receptor activity on mature oligodendrocytes attenuates loss of myelinated axons in autoimmune neuroinflammation.

Glutamate dysregulation occurs in multiple sclerosis (MS), but whether excitotoxic mechanisms in mature oligodendrocytes contribute to demyelination and axonal injury is unexplored. Although current treatments modulate the immune system, long-term disability ensues, highlighting the need for neuroprotection. Glutamate is elevated before T2-visible white matter lesions appear in MS. We previously reported that myelin-reactive T cells provoke microglia to release glutamate from the system xc - transporter promoting myelin degradation in experimental autoimmune encephalomyelitis (EAE). Here, we explore the target for glutamate in mature oligodendrocytes. Most glutamate-stimulated calcium influx into oligodendrocyte somas is AMPA receptor (AMPAR)-mediated, and genetic deletion of AMPAR subunit GluA4 decreased intracellular calcium responses. Inducible deletion of GluA4 on mature oligodendrocytes attenuated EAE and loss of myelinated axons was selectively reduced compared to unmyelinated axons. These data link AMPAR signaling in mature oligodendrocytes to the pathophysiology of myelinated axons, demonstrating glutamate regulation as a potential neuroprotective strategy in MS.