Allosteric Modulators Of AMPA Receptors Research Articles

Ampakines increase diaphragm activation following mid-cervical contusion injury in rats

Ampakines are positive allosteric modulators of AMPA receptors. We hypothesized that low-dose ampakine treatment increases diaphragm electromyogram (EMG) activity after mid-cervical contusion injury in rats. Adult male and female Sprague Dawley rats were implanted with in-dwelling bilateral diaphragm EMG electrodes. Rats received a 150 kDyn C4 unilateral contusion (C4Ct). At 4- and 14-days following C4Ct, rats were given an intravenous bolus of ampakine CX717 (5 mg/kg, n = 10) or vehicle (2-hydroxypropyl-beta-cyclodextrin; HPCD; n = 10). Diaphragm EMG was recorded while breathing was assessed using whole-body plethysmography. At 4-days, ampakine administration caused an immediate and sustained increase in bilateral peak inspiratory diaphragm EMG bursting and ventilation. The vehicle had no impact on EMG bursting. CX717 treated rats were able to increase EMG activity during a respiratory challenge to a greater extent vs. vehicle treated. Rats showed a considerable degree of spontaneous recovery of EMG bursting by 14 days, and the impact of CX717 delivery was blunted as compared to 4-days. Direct recordings from the phrenic nerve at 21–24 days following C4Ct confirmed that ampakines stimulated bilateral phrenic neural output in injured rats. We conclude that low-dose intravenous treatment with a low-impact ampakine can enhance diaphragm activation shortly following mid-cervical contusion injury, when deficits in diaphragm activation are prominent.

Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%–25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones.Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

Acute ampakines increase voiding function and coordination in a rat model of SCI.

Neurogenic bladder dysfunction causes urological complications and reduces the quality of life in persons with spinal cord injury (SCI). Glutamatergic signaling via AMPA receptors is fundamentally important to the neural circuits controlling bladder voiding. Ampakines are positive allosteric modulators of AMPA receptors that can enhance the function of glutamatergic neural circuits after SCI. We hypothesized that ampakines can acutely stimulate bladder voiding that has been impaired due to thoracic contusion SCI. Adult female Sprague-Dawley rats received a unilateral contusion of the T9 spinal cord (n = 10). Bladder function (cystometry) and coordination with the external urethral sphincter (EUS) were assessed 5 d post-SCI under urethane anesthesia. Data were compared to responses in spinal-intact rats (n = 8). The 'low-impact' ampakine CX1739 (5, 10, or 15 mg/kg) or vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was administered intravenously. The HPCD vehicle had no discernible impact on voiding. In contrast, following CX1739, the pressure threshold for inducing bladder contraction, voided volume, and the interval between bladder contractions were significantly reduced. These responses occurred in a dose-dependent manner. We conclude that modulating AMPA receptor function using ampakines can rapidly improve bladder-voiding capability at subacute time points following contusion SCI. These results may provide a new and translatable method for therapeutic targeting of bladder dysfunction acutely after SCI.

Acute ampakines increase voiding function and coordination in a rat model of SCI

Neurogenic bladder dysfunction causes urological complications and reduces the quality of life in persons with spinal cord injury (SCI). Glutamatergic signaling via AMPA receptors is fundamentally important to the neural circuits controlling bladder voiding. Ampakines are positive allosteric modulators of AMPA receptors that can enhance the function of glutamatergic neural circuits after SCI. We hypothesized that ampakines can acutely stimulate bladder voiding that has been impaired due to thoracic contusion SCI. Adult female Sprague–Dawley rats received a unilateral contusion of the T9 spinal cord (n = 10). Bladder function (cystometry) and coordination with the external urethral sphincter (EUS) were assessed 5 d post-SCI under urethane anesthesia. Data were compared to responses in spinal-intact rats (n = 8). The ‘low-impact’ ampakine CX1739 (5, 10, or 15 mg/kg) or vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was administered intravenously. The HPCD vehicle had no discernible impact on voiding. In contrast, following CX1739, the pressure threshold for inducing bladder contraction, voided volume, and the interval between bladder contractions were significantly reduced. These responses occurred in a dose-dependent manner. We conclude that modulating AMPA receptor function using ampakines can rapidly improve bladder-voiding capability at subacute time points following contusion SCI. These results may provide a new and translatable method for therapeutic targeting of bladder dysfunction acutely after SCI.

Positive modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors differentially alters spatial learning and memory in juvenile rats younger and older than three weeks.

Remarkable performance improvements occur at the end of the third postnatal week in rodents tested in various tasks that require navigation according to spatial context. While alterations in hippocampal function at least partially subserve this cognitive advancement, physiological explanations remain incomplete. Previously, we discovered that developmental modifications to hippocampal glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in juvenile rats was related to more mature spontaneous alternation behavior in a symmetrical Y-maze. Moreover, a positive allosteric modulator of AMPA receptors enabled immature rats to alternate at rates seen in older animals, suggesting an excitatory synaptic limitation to hippocampal maturation. We then validated the Barnes maze for juvenile rats in order to test the effects of positive AMPA receptor modulation on a goal-directed spatial memory task. Here we report the effects of the AMPA receptor modulator, CX614, on spatial learning and memory in the Barnes maze. Similar to our prior report, animals just over 3 weeks of age display substantial improvements in learning and memory performance parameters compared to animals just under 3 weeks of age. A moderate dose of CX614 enabled immature animals to move more directly to the goal location, but only after 1 day of training. This performance improvement was observed on the second day of training with drug delivery or during a memory probe trial performed without drug delivery after the second day of training. Higher doses created more search errors, especially in more mature animals. Overall, CX614 provided modest performance benefits for immature rats in a goal-directed spatial memory task.

5-Nitroisoxazoles in SNAr Reactions: A Novel Chemo- and Regioselective Approach to Isoxazole-Based Bivalent Ligands of AMPA Receptors.

An efficient regioselective approach to novel functionalized bis(isoxazoles) with a variety of aromatic and aliphatic linkers was elaborated, based on the heterocyclization reaction of electrophilic alkenes under the treatment with tetranitromethane-triethylamine complex affording 3-EWG-5-nitroisoxazoles. The subsequent SNAr reactions of 5-nitroisoxazoles with various O,O-, N,N- and S,S-bis(nucleophiles) provide a wide range of bis(isoxazole) derivatives in good isolated yields. Employing an elaborated method, a series of novel bis(3-EWG-isoxazoles) as the promising allosteric modulators of AMPA receptors were designed and synthesized. The effect of the compounds on the kainate-induced currents was studied in the patch clamp experiments, revealing modulator properties for several of them. The best positive modulator potency was found for dimethyl 5,5'-(ethane-1,2-diylbis(sulfanediyl))bis(isoxazole-3-carboxylate), which potentiated the kainate-induced currents in a wide concentration range (10-12-10-6 M) with maximum potentiation of 77% at 10-10 M. The results were rationalized using molecular docking and molecular dynamics simulations of modulator complexes with the dimeric ligand-binding domain of the GluA2 AMPA receptor. The predicted physicochemical, ADMET, and PAINS properties confirmed that the AMPA receptor modulators based on the bis(isoxazole) scaffold may serve as potential lead compounds for the development of neuroprotective drugs.

Knockdown of SIK3 in the CA1 Region can Reduce Seizure Susceptibility in Mice by Inhibiting Decreases in GABAAR α1 Expression.

Imbalance between excitation and inhibition is an important cause of epilepsy. Salt-inducible kinase 1 (SIK1) gene mutation can cause epilepsy. In this study, we first found that the expression of SIK3 is increased after epilepsy. Furthermore, the role of SIK3 in epilepsy was explored. In cultured hippocampal neurons, we used Pterosin B, a selective SIK3 inhibitor that can inhibit epileptiform discharges induced by the convulsant drug cyclothiazide (a positive allosteric modulator of AMPA receptors, CTZ). Knockdown of SIK3 inhibited epileptiform discharges and increased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). In mice, knockdown of SIK3 reduced epilepsy susceptibility in a pentylenetetrazole (a GABAA receptor antagonist, PTZ) acute kindling experiment and increased the expression of GABAA receptor α1. In conclusion, our results suggest that blockade or knockdown of SIK3 can inhibit epileptiform discharges and that SIK3 has the potential to be a novel target for epilepsy treatment.

New Allosteric Modulators of AMPA Receptors: Synthesis and Study of Their Functional Activity by Radioligand-Receptor Binding Analysis.

The synthetic approaches to three new AMPA receptor modulators-derivatives of 1,11-dimethyl-3,6,9-triazatricyclo[7.3.1.13,11]tetradecane-4,8,12-trione-had been developed and all steps of synthesis were optimized. The structures of the compounds contain tricyclic cage and indane fragments necessary for binding with the target receptor. Their physiological activity was studied by radioligand-receptor binding analysis using [3H]PAM-43 as a reference ligand, which is a highly potent positive allosteric modulator of AMPA receptors. The results of radioligand-binding studies indicated the high potency of two synthesized compounds to bind with the same targets as positive allosteric modulator PAM-43 (at least on AMPA receptors). We suggest that the Glu-dependent specific binding site of [3H]PAM-43 or the receptor containing this site may be one of the targets of the new compounds. We also suggest that enhanced radioligand binding may indicate the existence of synergistic effects of compounds 11b and 11c with respect to PAM-43 binding to the targets. At the same time, these compounds may not compete directly with PAM-43 for its specific binding sites but bind to other specific sites of this biotarget, changing its conformation and thereby causing a synergistic effect of cooperative interaction. It can be expected that the newly synthesized compounds will also have pronounced effects on the glutamatergic system of the mammalian brain.

Piracetam and Ginkgo biloba in the treatment of residual cognitive symptoms after the discontinuation of benzodiazepines in long-term users; case series and review of the literature

IntroductionLong-term benzodiazepine use is common although not routinely recommended. While their dependence potential is notorious, cognitive problems due to their chronic use have only recently been intensively studied. Residual cognitive symptoms may linger months after successful benzodiazepine discontinuation and significantly limit optimal functional outcome in abstinent individuals.ObjectivesWe present a series of cases, a 41-year-old man and a 33-year-old woman who successfully completed a ten-week benzodiazepine-dependence rehabilitation program at our hospital, including successful tapering and post-withdrawal integrative therapy. At subsequent monthly outpatient check-ups, abstinence was confirmed (including toxicology testing) and there was no residual anxiety or mood symptomatology. Still, patients complained of their suboptimal functioning due to residual cognitive problems, especially trouble concentrating and short-memory deficits, which were objectively confirmed via psychometric tests.MethodsDuring continuous outpatient treatment (monthly controls), patients were clinically and neurocognitively evaluated. They were reluctant to try conventional psychopharmacology agents but were open to supplement therapy. After thorough literature review, a trial of piracetam and Ginko biloba extract was suggested. Piracetam, a positive allosteric modulator of AMPA-receptors, has been used in the treatment of vascular neurocognitive changes in the elderly. Bilobalides from G. biloba, act as atypical antagonists of GABA-A receptors and were found to exert neuroprotective effects in animal models, without epileptogenic or anxiogenic risks.ResultsPatients were recommended piracetam (2.4 g a day) in divided doses and a once-daily dose of G. biloba extract (240 mg a day), in a standardized form containing 3.2% of bilobalides. No side effects were noticed. At subsequent monthly checkups, patients reported fewer cognitive problems and better everyday functioning. Neurocognitive testing confirmed subjective findings, with positive changes in all areas, but especially so in verbal memory, which may be due to this specific pharmacological combination. At the end of the observation period of sixth months, piracetam was gradually discontinued while patients were given free choice whether to continue with G. biloba supplementation.ConclusionsAccording to our findings, piracetam and G. biloba extract may prove beneficial in the treatment of residual cognitive symptoms after benzodiazepine discontinuation, in long-term benzodiazepine users. Other treatments, focusing not only on modulation of glutamate and GABA, but also on other pathways should be evaluated. Further clinical studies are warranted.Disclosure of InterestNone Declared

Novel Nanomolar Allosteric Modulators of AMPA Receptor of Bis(pyrimidine) Series: Synthesis, Biotesting and SAR Analysis

Positive allosteric modulators (PAMs) of AMPA receptors represent attractive candidates for the development of drugs for the treatment of cognitive and neurodegenerative disorders. Dimeric molecules have been reported to have an especially potent modulating effect, due to the U-shaped form of the AMPA receptor's allosteric binding site. In the present work, novel bis(pyrimidines) were studied as AMPA receptor modulators. A convenient and flexible preparative approach to bis(pyrimidines) containing a hydroquinone linker was elaborated, and a series of derivatives with varied substituents was obtained. The compounds were examined in the patch clamp experiments for their influence on the kainate-induced currents, and 10 of them were found to have potentiating properties. The best potency was found for 2-methyl-4-(4-((2-methyl-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)phenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine, which potentiated the kainate-induced currents by up to 77% in all tested concentrations (10-12-10-6 M). The results were rationalized via the modeling of modulator complexes with the dimeric ligand binding domain of the GluA2 AMPA receptor, using molecular docking and molecular dynamics simulation. The prediction of ADMET, physicochemical, and PAINS properties of the studied bis(pyrimidines) confirmed that PAMs of this type may act as the potential lead compounds for the development of neuroprotective drugs.

Ampakine CX717 Stimulates Diaphragm Activity following Mid‐cervical Spinal Contusion Injury

Cervical spinal cord injuries result in significant respiratory compromise and there is a need to develop therapies to counter respiratory insufficiency in this patient population. Ampakines are positive allosteric modulators of AMPA receptors, and can stimulate breathing during states of hypoventilation, such as following opioid overdose or in neuromuscular disorders. Our laboratory has previously shown that acute treatment with ampakine CX717 can stimulate phrenic activity in spinally injured animals under urethane anesthesia. Recently, we extended these findings by showing that a low dose of CX717 was sufficient to enhance diaphragm electromyography (EMG) activity and tidal volume in unanaesthetized animals with a C2 hemisection injury. The next step in the translational pathway, and the goal of the current study, was to test the efficacy of ampakines in a model of contusion injury that is more similar to the injuries typically experienced by humans. The C4 contusion injury used in the current study results in direct phrenic motor neuron loss and significant damage of the spinal pathways innervating respiratory motor pools. This model is also associated with strong spontaneous recovery of diaphragm function, such that pronounced deficits are likely to be detected only in the initial week following injury. Animals were implanted with in‐dwelling bilateral diaphragm EMG electrodes and ventilation was measured using whole body plethysmography. At 4 and 14 days following a 150 kDy C4 unilateral contusion injury (C4Ct), male and female rats were given a single intravenous bolus of CX717 (5 mg/kg, n=4) or vehicle (2‐hydroxypropyl‐beta‐cyclodextrin; HPCD; n=4). At the 4‐day time point, infusion of CX717 increased peak diaphragm EMG (recorded ipsilateral to C4Ct) by 40‐50% when compared to vehicle group. Respiratory rate was also elevated in the CX717 group by 80% post‐infusion, indicating a likely supraspinal mechanism of the impact of ampakines. In the CX717 group, tidal volume was increased by 25±13% during infusion, 20±16% at 5 min post infusion and returned to baseline levels by 15 mins post CX717 infusion. There was no change in tidal volume in the vehicle group. During an acute respiratory challenge with hypoxia (10% O2), CX717 treated animals were able to increase diaphragm EMG (100±15%) to a greater extent as compared to vehicle (50 ± 23%, P=0.004). Similarly, during a combined hypoxia‐hypercapnia challenge (10% O2, 7% CO2), CX717 treated animals displayed an increased diaphragm EMG response (200±44%) compared to vehicle (100 ± 55%, P=0.0144). In contrast to the 4‐day data, there was no discernable impact of ampakines on baseline or challenged breathing at 14 days post C4Ct. This is perhaps not surprising because prior work establishes that in this injury model, robust compensation has occurred after 2 weeks, and deficits in diaphragm activation are difficult to detect. In summary, our data indicate that low dose, low impact ampakine treatment can increase diaphragm muscle output at an acute timepoint following mid‐cervical contusion type injuries. These studies pave the path for future experiments to test if ampakine treatment can be combined with rehabilitation techniques to further enhance the therapeutic effect of ampakines in this injury model.

Ampakine‐Hypoxia Induced Phrenic Motor Facilitation is Not Prevented by Intrathecal Methysergide

Hypoxia‐induced sustained increases in phrenic nerve burst amplitude lasting beyond the hypoxic stimulus are generally accepted to require repeated bouts of hypoxia (i.e., acute intermittent hypoxia, AIH). However, our laboratory recently reported that pretreatment with a drug (ampakine) which acts as a positive allosteric modulator of AMPA receptors lowered the number of hypoxic exposures required to trigger sustained phrenic motor facilitation. Our working hypothesis for the current study was that the combination of ampakine and hypoxia activates the same serotonin (5‐HT) dependent cellular mechanisms that are well established for AIH‐induced phrenic long‐term facilitation following repeated bouts of moderate hypoxia. Phrenic nerve output was recorded from vagotomized, mechanically ventilated, adult male Sprague‐Dawley rats under urethane‐anesthesia. A broad‐spectrum 5‐HT receptor antagonist (methysergide, 20mM, dissolved in DMSO) was delivered intrathecally over the C4 spinal dorsal surface. At least 15 min of stable phrenic burst amplitude was recorded post methysergide delivery. After a stable baseline (BL) was established, intravenous (i.v.) ampakine (CX717, 15mg/kg) was followed by a single episode of hypoxia (arterial partial pressure of O2 = 42±9mmHg). Phrenic nerve bursting was recorded for 60 min after hypoxia. Initial results indicate that intrathecal methysergide does not prevent the ampakine‐hypoxia induced phrenic motor facilitation as evidenced by a significant increase in burst amplitude at 60 min (89±54% BL, n=7). To verify the efficacy of intrathecal drug delivery method, additional experiments were conducted. Specifically, we determined if intrathecal methysergide prevented the phrenic long‐term facilitation induced by AIH, as has been shown by several prior investigations. These experiments used the same preparation, and AIH consisted of three‐5 min episodes of hypoxia with 5‐min intervals of baseline O2conditions. Intrathecal methysergide blocked pLTF (60 min post‐AIH, phrenic burst amplitude = ‐2±31% BL, n=3), but intrathecal delivery of the DMSO vehicle did not (60 min: 94±28% BL, n=3). Collectively, these results do not support our hypothesis that phrenic motor facilitation induced by the pairing of ampakine with a single bout of hypoxia is dependent on serotonin receptor activation.

Automated Classification of Whole Body Plethysmography Waveforms to Quantify Breathing Patterns

Whole body plethysmography (WBP) monitors respiratory rate and depth but conventional analysis fails to capture the diversity of waveforms. Our first purpose was to develop a waveform cluster analysis method for quantifying dynamic changes in respiratory waveforms. WBP data, from adult Sprague-Dawley rats, were sorted into time domains and principle component analysis was used for hierarchical clustering. The clustering method effectively sorted waveforms into categories including sniffing, tidal breaths of varying duration, and augmented breaths (sighs). We next used this clustering method to quantify breathing after opioid (fentanyl) overdose and treatment with ampakine CX1942, an allosteric modulator of AMPA receptors. Fentanyl caused the expected decrease in breathing, but our cluster analysis revealed changes in the temporal appearance of inspiratory efforts. Ampakine CX1942 treatment shifted respiratory waveforms toward baseline values. We conclude that this method allows for rapid assessment of breathing patterns across extended data recordings. Expanding analyses to include larger portions of recorded WBP data may provide insight on how breathing is affected by disease or therapy.

Ampakines enhance phrenic nerve output in a ZFN rat model of Pompe Disease

Pompe disease (PD) is caused by mutations in the gene encoding acid-α-glucosidase (GAA), an enzyme that degrades lysosomal glycogen. The resultant glycogen accumulation is associated with neurodegeneration, cardiac deficits, and impaired phrenic motoneuron and diaphragm activation. Cardiorespiratory failure is the leading cause of death in early-onset PD patients. We previously reported that a drug which acts as a positive allosteric modulator of AMPA receptors, ampakine CX717, could stimulate phrenic motor output and increase minute ventilation in a mouse model of Pompe disease (Gaa-/-). We recently created a new rat model of PD that better recapitulates the clinical phenotype of early onset PD, called the Zinc Finger Nuclease (ZFN) KO model. Phrenic nerve recordings were performed on urethane anesthetized ZFN KO rats of both sexes, and age-matched wild type (WT) controls during a baseline normoxic period, followed by a brief exposure to hypoxia (PaO2 ~ 30mmHg) and a maximal respiratory challenge with combined hypoxia-hypercapnia. Recordings were done before and after treatment with ampakine CX717 (15mg/kg) or its vehicle control (2-Hydroxypropyl)-β-cyclodextrin. Our goal was to build upon the prior data from the Gaa-/-mouse, by using the ZFN KO model to test the hypothesis that in addition to stimulating basal or “eupneic” phrenic output, ampakine CX717 could also increase the ability to respond to acute respiratory challenge. We also aimed to determine if the extent of phrenic motor deficits were different between male and female ZFN KO rats. The data confirm that both male and female ZFN KO rats exhibit cardiac irregularities and phrenic motor deficits when compared to WT controls. However, a sex difference was apparent as male ZFN KO rats exhibit greater phrenic motor deficits and blunted responses to gas challenges compared to female ZFN KO rats. Ampakine treatment enhanced phrenic output and improved the ability to respond to respiratory challenge in both sexes of the ZNF rat model of PD. We conclude that 1) sex differences are present in the phrenic motor phenotype of ZFN KO rats, 2) the ability of low dose CX717to stimulate phrenic motor output in PD is reproducible across two species, and 3) low dose CX717 can enhance the ability to respond to respiratory challenge in PD rats These data suggest that ampakines may serve as a viable adjunctive therapy for respiratory insufficiency in PD patients.

Characterization of a New Positive Allosteric Modulator of AMPA Receptors - PAM-43: Specific Binding of the Ligand and its Ability to Potentiate AMPAR Currents.

Currently, the most dynamic areas in the glutamate receptor system neurobiology are the identification and development of positive allosteric modulators (PAMs) of glutamate ionotropic receptors. PAM-based drugs are of great interest as promising candidates for the treatment of neurological diseases, such as epilepsy, Alzheimer's disease, schizophrenia, etc. Understanding the molecular mechanisms underlying the biological action of natural and synthetic PAMs is a key point for modifying the original chemical compounds as well as for new drug design. We are trying to elaborate a system of molecular functional screening of ionotropic glutamate receptor probable PAMs. The system will be based on the radioligand - receptor method of analysis and will allow rapid quantification of new AMPAR probable PAMs molecular activity. We plan to use a tritiumlabeled analogue of recently elaborated ionotropic GluR probable PAM ([3H]PAM-43) as the main radioligand. Here, we characterized the specific binding of the ligand and its ability to potentiate ionotropic GluR currents. The existence of at least two different sites of [3H]PAM-43 specific binding has been shown. One of the above sites is glutamate-dependent and is characterized by higher affinity. "Patchclamp" technique showed the ability of PAM-43 to potentiate ionotropic GluR currents in rat cerebellar Purkinje neurons in a concentration-dependent manner. The possibility of using PAM-43 as a model compound to study different allosteric effects of potential regulatory drugs (AMPAR allosteric regulators) was shown. [3H]PAM-43 based screening system will allow rapid selection of new AMPAR probable PAM structures and quantification of their molecular activity.

Allosteric modulation of AMPA receptors counteracts Tau-related excitotoxic synaptic signaling and memory deficits in stress- and Aβ-evoked hippocampal pathology.

Despite considerable progress in the understanding of its neuropathology, Alzheimer's disease (AD) remains a complex disorder with no effective treatment that counteracts the memory deficits and the underlying synaptic malfunction triggered by the accumulation of amyloid beta (Aβ) and Tau protein. Mounting evidence supports a precipitating role for chronic environmental stress and glutamatergic excitotoxicity in AD, suggesting that targeting of glutamate receptor signaling may be a promising approach against both stress and AD pathologies. In light of the limited cognitive benefit of the direct antagonism of NMDA receptors in AD, we here focus on an alternative way to modify glutamatergic signaling through positive allosteric modulation of AMPA receptors, by the use of a PAM-AMPA compound. Using non-transgenic animal model of Aβ oligomer injection as well as the combined stress and Aβ i.c.v. infusion, we demonstrate that positive allosteric modulation of AMPA receptors by PAM-AMPA treatment reverted memory, but not mood, deficits. Furthermore, PAM-AMPA treatment reverted stress/Aβ-driven synaptic missorting of Tau and associated Fyn/GluN2B-driven excitotoxic synaptic signaling accompanied by recovery of neurotransmitter levels in the hippocampus. Our findings suggest that positive allosteric modulation of AMPA receptors restores synaptic integrity and cognitive performance in stress- and Aβ-evoked hippocampal pathology. As the prevalence of AD is increasing at an alarming rate, novel therapeutic targeting of glutamatergic signaling should be further explored against the early stages of AD synaptic malfunction with the goal of attenuating further synaptic damage before it becomes irreversible.

Impact of Ampakine CX‐717 on Diaphragm EMG Activity and Breathing in Non‐anesthetized Freely Behaving Rats

Glutamatergic neurotransmission is partly mediated by ionotropic AMPA receptors, and these receptors play an important role in the neural control of breathing. Ampakines are positive allosteric modulators of AMPA receptors. These drugs can stimulate breathing during hypo‐ventilated states, such as following an opioid overdose. Our laboratory has recently shown that acute exposure to an ampakine, CX717 (15 mg/kg), can acutely stimulate neural drive to the diaphragm in anesthetized but otherwise healthy rats. The goal of the current work is to evaluate the impact of ampakine on diaphragm muscle activation in freely moving non‐anesthetized rats. Ventilation and metabolic rate (VCO2) were measured using whole body plethysmography and diaphragm EMG was assessed using intramuscular electrodes. Spinally intact rats (n=2) were given a single intravenous bolus of CX717 at four months after implantation of diaphragm EMG electrodes. Delivering CX717 at 15 mg/kg had no detectable impact on VCO2, inspiratory tidal volume or diaphragm EMG burst amplitude. In addition, at 30 minutes following IV ampakine, both animals showed a robust increase in diaphragm EMG activity, tidal volume, and the ratio of ventilation VCO2 (VE/VCO2) during an acute hypoxic challenge (5 min, 10.5% inspired O2). Qualitatively, we observed a possible sedative effect as shown of lack of movement and exploratory behavior following ampakine exposure. Our preliminary conclusion is that ampakine CX717 at a dose of 15 mg/kg has little impact on breathing in the otherwise healthy rat. Ongoing studies are evaluating impact of the vehicle (2‐hydroxypropyl‐beta‐cyclodextrin) versus ampakine using a cross‐over study design in spinally intact rats and in rats with spinal cord injury.Support or Funding Information5 R01 HL139708 02 (DDF)F31 HL145831‐01 (MDS)

Hippocampal gamma rhythms during Y-maze navigation in the juvenile rat.

The neurobiology of postnatal hippocampal development in rodents is receiving increased attention as a means to address neurodevelopmental questions and to better understand the neural code(s) for spatial navigation in adulthood. We previously showed that spontaneous alternation (SA) in a Y-maze, which emerges at the end of the third postnatal week, was related to changes in fast glutamatergic synaptic transmission. In adults, oscillations in the hippocampal local field potential (LFP) (i.e., theta, 4-12 Hz; slow gamma, 25-55 Hz; and fast gamma, 65-100 Hz) have been shown to coordinate the activity of spatially tuned cell types in the hippocampus during route planning and execution. Other investigators have shown that theta activity matures during the first month of life. However, relationships between developmental alterations in gamma oscillations and cognitive function have not been investigated in juveniles, and the impact of developmental changes in excitatory synaptic transmission on network activity remains unclear. We implanted rats at postnatal day 14 to record LFPs from the synaptic layer of area CA1 during Y-maze exploration at postnatal Days 18, 19, 23, and 24. The positive allosteric modulator of AMPA receptors, CX614, or vehicle was administered prior to each test. We found that slow gamma peak frequency, but not peak power, remained constant across this age range. Fast gamma event rate increased with increasing age, while peak frequency decreased. AMPA receptor modulation impacted slow and fast gamma differently at different ages. Furthermore, gamma events showed relationships with novelty and movement speed that differed based on gamma sub-band (slow vs. fast). These results support the presence of environmentally-driven gamma oscillations in hippocampal network activity prior to the end of the third postnatal week of development. Differential refinement of slow and fast gamma occurring across the subsequent week supports involvement in the emergence of spatial navigation ability.

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs.

New positive allosteric modulator of AMPA-receptors: in vitro and in vivo studies

A new derivative of 3,7-diazabicyclo[3.3.1]nonane was studied in electrophysiological experiments, which showed high activity as a positive allosteric modulator of AMPA receptors of the CNS. In doses of 0.01 mg/kg this compound significantly improved the memory of experimental animals disturbed by maximal electric shock. The results indicate that this compound is a promising candidate for preclinical and clinical studies as a treatment for a number of psycho-neurological diseases.