Ionotropic Glutamate Receptor Agonists Research Articles

Familial Alzheimer's Disease Neurons Bearing Mutations in PSEN1 Display Increased Calcium Responses to AMPA as an Early Calcium Dysregulation Phenotype.

Familial Alzheimer's disease (FAD) can be caused by mutations in PSEN1 that encode presenilin-1, a component of the gamma-secretase complex that cleaves amyloid precursor protein. Alterations in calcium (Ca2+) homeostasis and glutamate signaling are implicated in the pathogenesis of FAD; however, it has been difficult to assess in humans whether or not these phenotypes are the result of amyloid or tau pathology. This study aimed to assess the early calcium and glutamate phenotypes of FAD by measuring the Ca2+ response of induced pluripotent stem cell (iPSC)-derived neurons bearing PSEN1 mutations to glutamate and the ionotropic glutamate receptor agonists NMDA, AMPA, and kainate compared to isogenic control and healthy lines. The data show that in early neurons, even in the absence of amyloid and tau phenotypes, FAD neurons exhibit increased Ca2+ responses to glutamate and AMPA, but not NMDA or kainate. Together, this suggests that PSEN1 mutations alter Ca2+ and glutamate signaling as an early phenotype of FAD.

FRI275 Glutamate Receptor Agonists Stimulate Kisspeptin Neurons In The Arcuate Nucleus Of The Hypothalamus

Abstract Disclosure: R. Bearss: None. R. Piet: None. KNDy neurons co-express kisspeptin, neurokinin B (NKB), dynorphin A (Dyn A), and glutamate. These neurons, located in the arcuate nucleus of the hypothalamus, drive pulsatile gonadotropin releasing hormone (GnRH) secretion. This eventually promotes pulsatile gonadotropin hormone release from the anterior pituitary, important for fertility in both sexes. The current hypothesized mechanism for pulse generation involves KNDy neuron interconnectivity and reciprocal regulation through the combined actions of NKB, DynA and glutamate to coordinate KNDy neuron activity. There is evidence that KNDy neurons express NKB, Dyn A and glutamate receptors and that NKB stimulates KNDy neuron electrical activity while Dyn A suppresses it. There is also evidence that ionotropic glutamate receptor antagonists can inhibit KNDy neuron coordinated activity and pulsatile gonadotropin hormone release, indicating that glutamatergic neurotransmission plays a significant role in activity coordination. The purpose of the following experiments was to investigate the response of KNDy neurons to varied glutamate receptor activation. We used calcium imaging to monitor the activity of KNDy neurons in brain slices. We crossed Kiss1-Cre mice—expressing Cre recombinase (Cre) in kisspeptin neurons—with Cre-dependent GCaMP6f mice to generate Kiss1-Cre x GCaMP6f mice that express GCaMP6f in kisspeptin neurons. GCaMP6f fluorescence increases when binding to intracellular calcium and can, thus, be used as an inferential marker of electrical activity. Coronal brain slices (200 µm thickness) obtained from male Kiss1-cre x GCaMP6f mice and containing the arcuate nucleus were placed in a recording chamber under an epifluorescence microscope. KNDy neurons were exposed for 2 minutes to a bath application of either the ionotropic glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 10µM), the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA, 50µM), or the group 1 metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 50µM). AMPA, NMDA, and DHPG increased KNDy neuron fluorescence by 7.8±0.5% (n=113 in 7 slices from 6 mice), 7.5±0.5% (n=154 in 6 slices from 5 mice), and 6.1±0.7% (n=159 in 7 slices from 5 mice) respectively. All cells that were excited by NMDA or DHPG were also excited by AMPA. These experiments provide evidence that activation of ionotropic AMPA and NMDA receptors as well as group 1 mGluRs stimulate KNDy neuron activity. Further investigation into the precise role of individual glutamate receptor subtypes on KNDy neuron coordinated activity is needed. Presentation: Friday, June 16, 2023

The substantia nigra modulates proximal colon tone and motility in a vagally-dependent manner in the rat.

A monosynaptic pathway connects the substantia nigra pars compacta (SNpc) to neurons of the dorsal motor nucleus of the vagus (DMV). This monosynaptic pathway modulates the vagal control of gastric motility. It is not known, however, whether this nigro-vagal pathway also modulates the tone and motility of the proximal colon. In rats, microinjection of retrograde tracers in the proximal colon and of anterograde tracers in SNpc showed that bilaterally labelled colonic-projecting neurons in the DMV received inputs from SNpc neurons. Microinjections of the ionotropic glutamate receptor agonist, NMDA, in the SNpc increased proximal colonic motility and tone, as measured via a strain gauge aligned with the colonic circular smooth muscle; the motility increase was inhibited by acute subdiaphragmatic vagotomy. Upon transfection of SNpc with pAAV-hSyn-hM3D(Gq)-mCherry, chemogenetic activation of nigro-vagal nerve terminals by brainstem application of clozapine-N-oxide increased the firing rate of DMV neurons and proximal colon motility; both responses were abolished by brainstem pretreatment with the dopaminergic D1-like antagonist SCH23390. Chemogenetic inhibition of nigro-vagal nerve terminals following SNpc transfection with pAAV-hSyn-hM4D(Gi)-mCherry decreased the firing rate of DMV neurons and inhibited proximal colon motility. These data suggest that a nigro-vagal pathway modulates activity of the proximal colon motility tonically via a discrete dopaminergic synapse in a manner dependent on vagal efferent nerve activity. Impairment of this nigro-vagal pathway may contribute to the severely reduced colonic transit and prominent constipation observed in both patients and animal models of parkinsonism. KEY POINTS: Substantia nigra pars compacta (SNpc) neurons are connected to the dorsal motor nucleus of the vagus (DMV) neurons via a presumed direct pathway. Brainstem neurons in the lateral DMV innervate the proximal colon. Colonic-projecting DMV neurons receive inputs from neurons of the SNpc. The nigro-vagal pathway modulates tone and motility of the proximal colon via D1-like receptors in the DMV. The present study provides the mechanistic basis for explaining how SNpc alterations may lead to a high rate of constipation in patients with Parkinson's Disease.

Total synthesis of (-)-domoic acid, a potent ionotropic glutamate receptor agonist and the key compound in oceanic harmful algal blooms.

The stereo-controlled total synthesis of (-)-domoic acid is described. The critical construction of the C1'-C2' Z-configuration was accomplished by taking advantage of an unsaturated lactam structure. The side chain fragment was introduced in the final stages of synthesis through a modified Julia-Kocieński reaction, aiming for its efficient derivatization.

Willardiine and Its Synthetic Analogues: Biological Aspects and Implications in Peptide Chemistry of This Nucleobase Amino Acid

Willardiine is a nonprotein amino acid containing uracil, and thus classified as nucleobase amino acid or nucleoamino acid, that together with isowillardiine forms the family of uracilylalanines isolated more than six decades ago in higher plants. Willardiine acts as a partial agonist of ionotropic glutamate receptors and more in particular it agonizes the non-N-methyl-D-aspartate (non-NMDA) receptors of L-glutamate: ie. the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate receptors. Several analogues and derivatives of willardiine have been synthesised in the laboratory in the last decades and these compounds show different binding affinities for the non-NMDA receptors. More in detail, the willardiine analogues have been employed not only in the investigation of the structure of AMPA and kainate receptors, but also to evaluate the effects of receptor activation in the various brain regions. Remarkably, there are a number of neurological diseases determined by alterations in glutamate signaling, and thus, ligands for AMPA and kainate receptors deserve attention as potential neurodrugs. In fact, similar to willardiine its analogues often act as agonists of AMPA and kainate receptors. A particular importance should be recognized to willardiine and its thymine-based analogue AlaT also in the peptide chemistry field. In fact, besides the naturally-occurring short nucleopeptides isolated from plant sources, there are different examples in which this class of nucleoamino acids was investigated for nucleopeptide development. The applications are various ranging from the realization of nucleopeptide/DNA chimeras for diagnostic applications, and nucleoamino acid derivatization of proteins for facilitating protein-nucleic acid interaction, to nucleopeptide-nucleopeptide molecular recognition for nanotechnological applications. All the above aspects on both chemistry and biotechnological applications of willardine/willardine-analogues and nucleopeptide will be reviewed in this work.

Low-Intensity Ultrasound Decreases Ischemia-Induced Edema by Inhibiting N-Methyl-d-Aspartic Acid Receptors.

We have previously shown that low-intensity ultrasound (LIUS), a noninvasive mechanical stimulus, inhibits brain edema formation induced by oxygen and glucose deprivation (OGD) or treatment with glutamate, a mediator of OGD-induced edema, in acute rat hippocampal slice model in vitro. In this study, we treated the rat hippocampal slices with N-methyl-d-aspartic acid (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) to determine whether these different glutamate receptor agonists induce edema. The hippocampal slices were then either sonicated with LIUS or treated with N-methyl-d-aspartic acid receptor (NMDAR) antagonists, namely, MK-801 and ketamine, and observed their effects on edema formation. We observed that treatment with NMDA, an agonist of ionotropic glutamate receptors, induced brain edema at similar degrees compared with that induced by OGD. However, treatment with DHPG, an agonist of metabotropic glutamate receptors, did not significantly induce brain edema. Treatment with the NMDAR antagonists MK-801 or ketamine efficiently prevented brain edema formation by both OGD and NMDA in a concentration-dependent manner. N-Methyl-d-aspartic acid-induced brain edema was alleviated by LIUS in an intensity-dependent manner when ultrasound was administered at 30, 50, or 100 mW/cm2 for 20 minutes before the induction of the edema. Furthermore, LIUS reduced OGD- and NMDA-induced phosphorylation of NMDARs at Y1325. These results suggest that LIUS can inhibit OGD- or NMDA-induced NMDAR activation by preventing NMDAR phosphorylation, thereby reducing a subsequent brain edema formation. The mechanisms by which LIUS inhibits NMDAR phosphorylation need further investigation.

The Effect of Rosmarinic Acid on Apoptosis and nNOS Immunoreactivity Following Intrahippocampal Kainic Acid Injections in Rats.

Introduction:Kainic Acid (KA) is an ionotropic glutamate receptor agonist. KA can induce neuronal overactivity and excitotoxicity. Rosmarinic Acid (RA) is a natural polyphenolic compound with antioxidant, anti-apoptotic, anti-neurodegenerative, and anti-inflammatory properties. This study aimed to assess the effect of RA on apoptosis, nNOS-positive neurons number, as well as Mitogen-Activated Protein Kinase (MAPK) and Cyclooxygenase-2 (COX-2) immunoreactivity, following intrahippocampal Kainic acid injection in rats.Methods:The study rats were randomly assigned to three groups of sham, KA (KA was injected into the right side of the hippocampus) and KA+RA (a dose of 10 mg/kg/day through a gavage needle for one week before KA injection). Then, histopathological changes, including apoptosis [Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay], nNOS-positive neurons number, as well as COX-2 and MAPK immunoreactivity were evaluated in the hippocampus.Results:In the RA pretreated group, nNOS-positive neurons and TUNEL-positive cells were significantly reduced compared to the KA group (P<0.05). COX-2and MAPK immunoreactivity demonstrated no significant changes compared to the KA group. They indicated a significant higher reactivity for COX-2 (P<0.01) and MAPK (P<0.005) versus the sham group.Conclusion:RA had neuroprotective effects, compared to KA, through reduced apoptosis and nNOS-positive neurons, but not MAPK and COX-2.

Vinpocetine protects inner retinal neurons with functional NMDA glutamate receptors against retinal ischemia

Retinal ischemia is involved in the pathogenesis of many major vision threatening diseases. Vinpocetine is a natural drug, which has a range of neuroprotective actions against retinal ischemia including modulating cation flow, improving metabolic activity and preventing apoptosis. The exact mechanism behind these actions remains unknown but may involve glutamate receptors, major components of the ischemic cascade. This study examined the effects of vinpocetine in association with specific ionotropic glutamate receptor agonists: N-methyl-D-aspartate (NMDA) and kainate. Vinpocetine's actions to improve cation channel permeability and cell marker immunoreactivity following ischemia appeared to be limited to NMDA activation with no changes observed following kainate stimulation. Vinpocetine's actions were lost in the presence of an NMDA receptor inhibitor further suggesting they may be secondary to NMDA receptor activation. NMDA receptor function was also necessary for vinpocetine's actions on glucose availability during ischemia but not lactate dehydrogenase (LDH) activity in the ischemic retina suggesting not all of vinpocetine's actions are linked to NMDA receptor function. These results may explain vinpocetine's effectiveness as a neuroprotective agent as the NMDA receptor is implicated in the pathogenesis of ischemia in a range of tissues of the central nervous system.

Harmful Effects of Neonatal Epileptic Seizures on Cerebrovascular Functions: Does Sex Matter?

There is a growing interest in the potential contribution of sex‐related variables to cerebrovascular functions in healthy and sick neonates. Epileptic seizures, the most common adverse neurological event in human newborns, may lead to cerebrovascular injury and abnormalities in cerebral blood flow regulation. Clinical studies suggest that male neonates are more susceptible to seizures, with a higher incidence of neurological complications. Cerebral vascular health is an important contributing factor to brain homeostasis and neuronal health. Newborn piglets provide a valuable and translationally relevant animal model for studying the cerebrovascular effects of epileptic seizures in the neonatal brain. This study in newborn pigs (2–5 days old) was designed to investigate whether sex‐related differences contribute to systemic parameters and cerebrovascular functions in: 1) control healthy animals and 2) piglets subjected to bicuculline‐induced epileptic seizures leading to prolonged cerebral vascular dysfunction during the delayed postictal period. After a 48‐h recovery period from epileptic seizures, postictal cerebrovascular functions were tested using intravital microscopy via the closed cranial window technique. We recorded the responses of small‐ and medium‐sized pial cerebral arterioles (50–100 μm) to a battery of vasodilator stimuli including: A) endothelium‐dependent hypercapnia and bradykinin; B) dual endothelium/astrocyte‐dependent stimuli glutamate, ionotropic glutamate receptor agonist quisqualic acid, and hemin; C) astrocyte‐dependent vasodilator adenosine diphosphate, and D) endothelium/astrocyte‐independent vasodilator sodium nitroprusside (SNP). We found no sex‐related differences in cardiovascular parameters (mean arterial blood pressure, heart rate), arterial blood gases, or core body temperature in either control or postictal groups of newborn piglets. A significant reduction (50–70%) in responses of pial arterioles to endothelium‐ and/or astrocyte‐dependent vasodilators was observed 48 h after seizures (delayed postictal period) in both male and female piglets. In piglets of both sexes, vasodilation to SNP was preserved during the delayed postictal period, suggesting that vascular smooth muscle contractility was not affected by seizures. An antioxidant and cytoprotective compound, the carbon‐monoxide (CO)‐releasing molecule CORM‐A1 (3 mg/kg i.p.) that was administered shortly before and/or during seizures, prevented postictal loss of cerebrovascular functions in both male and female piglets. Overall, we observed no sex‐specific differences in selected systemic cardiovascular parameters and cerebrovascular dilator functions in control and epileptic newborn pigs. In both male and female piglets, epileptic seizures lead to severe cerebral vascular dysfunction during the delayed postictal period. Antioxidant therapy with CORM‐A1 is neuroprotective in both male and female newborn pigs because it effectively prevents deleterious effects of epileptic seizures on cerebral blood flow regulation in a sex‐independent manner.Support or Funding InformationThe study was supported by NIH/NHLBI/NINDS.

Excessive activation of ionotropic glutamate receptors induces apoptotic hair-cell death independent of afferent and efferent innervation

Accumulation of excess glutamate plays a central role in eliciting the pathological events that follow intensely loud noise exposures and ischemia-reperfusion injury. Glutamate excitotoxicity has been characterized in cochlear nerve terminals, but much less is known about whether excess glutamate signaling also contributes to pathological changes in sensory hair cells. I therefore examined whether glutamate excitotoxicity damages hair cells in zebrafish larvae exposed to drugs that mimic excitotoxic trauma. Exposure to ionotropic glutamate receptor (iGluR) agonists, kainic acid (KA) or N-methyl-D-aspartate (NMDA), contributed to significant, progressive hair cell loss in zebrafish lateral-line organs. To examine whether hair-cell loss was a secondary effect of excitotoxic damage to innervating neurons, I exposed neurog1a morphants—fish whose hair-cell organs are devoid of afferent and efferent innervation—to KA or NMDA. Significant, dose-dependent hair-cell loss occurred in neurog1a morphants exposed to either agonist, and the loss was comparable to wild-type siblings. A survey of iGluR gene expression revealed AMPA-, Kainate-, and NMDA-type subunits are expressed in zebrafish hair cells. Finally, hair cells exposed to KA or NMDA appear to undergo apoptotic cell death. Cumulatively, these data reveal that excess glutamate signaling through iGluRs induces hair-cell death independent of damage to postsynaptic terminals.

The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca(2+)]i.

Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration ([Ca2+]i) increased in response to these glutamate agonists. The one exception was AMPA that increased [Ca2+]i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase [Ca2+]i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect.

Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGATflox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT−/− mice were the same as Cx57-VGAT+/+ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT−/− mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT−/− mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

Ameliorative potential of standardized fruit extract of Pterodon pubescens Benth on neuropathic pain in mice: Evidence for the mechanisms of action

Ethnopharmacological relevanceThe medicinal plant Pterodon pubescens Benth has been traditionally used for a long time to treat rheumatic diseases due to its anti-inflammatory and analgesic activities. The present study aims to evaluate the antinociceptive effect of ethanolic extract from P. pubescens fruits (EEPp) in a model of neuropathic pain in mice. Materials and methodsThe phytochemical analysis of EEPp was performed through GC–MS, HPLC and colorimetric analysis. The antinociceptive effects of EEPp (30–300mg/kg, i.g.) were evaluated on mechanical and thermal (cold or heat) hyperalgesia in neuropathic pain induced by partial sciatic nerve ligation (PSNL) in mice. We also investigated the effects of EEPp on the nociceptive response induced by intrathecal injection (i.t.) of ionotropic (AMPA, NMDA and kainate) and metabotropic (trans-ACPD) glutamate receptor agonists, proinflammatory cytokines such as IL-1β and TNF-α, as well as TRPV1 and TRPA1 agonists. In addition, we also investigated the safety profile of prolonged treatment with EEPp in mice. ResultsThe phytochemical analysis showed a higher amount terpenes, being nine sesquiterpenes and seven diterpenes with vouacapan skeletons, as well as a small amount of phenols and flavonoids. The exact mechanism by which EEPp promotes its antinociceptive effect is not yet fully understood, but its oral administration causes significant inhibition of glutamate-, kainate-, NMDA-, trans-ACPD-induced biting responses, as well as of proinflammatory cytokines (TNF-α and IL-1β) and TRPV1 and TRPA1 channels activators (capsaicin and cinnamaldehyde, respectively). These results may indicate, at least in part, some of the mechanisms that are involved in this effect. In particular, EEPp decreases neuropathic pain and clearly shows, for the first time, a thermal and mechanical hyperalgesia reduction in the model of partial sciatic nerve ligation (PSNL), without inducing tolerance. Furthermore, the prolonged treatment with EEPp (300mg/kg, i.g.) showed a cumulative effect over 24h, in the 15th day, after last treatment. In addition, the open-field test showed that doses up to 300mg/kg in both treatments, acute and/or prolonged, did not affect the motor activity of mice. Also, EEPp showed no toxicity according to the serum levels of the renal and hepatic injury indicators or observed macroscopic organs, after PSNL. ConclusionsTaken together, these results provide the first experimental evidence of the significant antinociceptive effect of EEPp on neuropathic pain without causing side effects, such as sedation or locomotor dysfunction. Moreover, these results appear to be mediated, at least in part, by the inhibition of glutamatergic receptors, TRPV1 and TRPA1 channels and proinflammatory cytokines. Thus, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant P. pubescens in the development of phytomedicines for the management of neuropathic pain.

Retraction: Radenovic L, Selakovic V. Mitochondrial superoxide production and MnSOD activity following exposure to an agonist and antagonists of ionotropic receptors in rat brain, Arch Biol Sci. 2005; 57(1):1-10, DOI:10.2298/ABS0501001R

This is a notice of retraction of the article: Mitochondrial superoxide production and MnSOD activity following exposure to an agonist and antagonists of ionotropic glutamate receptors in rat brain, published in the Archives of Biological Sciences in 2005, Vol. 57, Issue 1. The Editor-in-Chief has been informed that this paper plagiarizes an earlier paper: Radenovic L, Selakovic V, Kartelija G, Todorovic N, Nedeljkovic M. Differential effects of NMDA and AMPA/kainate receptor antagonists on superoxide production and MnSOD activity in rat brain following intrahippocampal injection. Brain Res Bull, 2004, 64(1):85-93. This claim is correct and almost the entire paper is a verbatim copy of the earlier one. After confirmation of this fact, the Editor-in-Chief of the Archives of Biological Sciences has decided to retract the paper immediately. We apologize to the readers of the journal that it took so many years to notice this error and to retract the paper. We request readers of the journal to directly get in touch with the editorial office and the editors of the journal for similar cases in the future, so that they can be handled promptly. &lt;br&gt;&lt;br&gt;&lt;font color="red"&gt;&lt;b&gt; Link to the retracted article &lt;u&gt;&lt;a href="http://dx.doi.org/10.2298/ABS0501001R"&gt;10.2298/ABS0501001R&lt;/a&gt;&lt;/b&gt;&lt;/u&gt;

Blood–Brain Barrier Permeability and Brain Uptake Mechanism of Kainic Acid and Dihydrokainic Acid

The glutamatergic neurotransmitter system is involved in important neurophysiological processes and thus constitutes a promising target for the treatment of neurological diseases. The two ionotropic glutamate receptor agonists kainic acid (KA) and dihydrokainic acid (DHK) have been used as research tools in various in vivo central nervous system disease models in rodents, as well as being templates in the design of novel ligands affecting the glutamatergic system. Both molecules are highly polar but yet capable of crossing the blood-brain barrier (BBB). We used an in situ rat brain perfusion technique to determine the brain uptake mechanism and permeability across the BBB. To determine KA and DHK concentrations in the rat brain, simple and rapid sample preparation and liquid chromatography mass spectrometer methods were developed. According to our results the BBB permeability of KA and DHK is low, 0.25 × 10(-6) and 0.28 × 10(-6) cm/s for KA and DHK, respectively. In addition, the brain uptake is mediated by passive diffusion, and not by active transport. Furthermore, the non-specific plasma and brain protein binding of KA and DHK was determined to be low, which means that the unbound drug volume of distribution in brain is also low. Therefore, even though the total KA and DHK concentrations in the brain are low after systemic dosing, the concentrations in the vicinity of the glutamate receptors are sufficient for their activation and thus the observed efficacy.

Citral: A monoterpene with prophylactic and therapeutic anti-nociceptive effects in experimental models of acute and chronic pain

Citral (3,7-dimethyl-2,6-octadienal) is an open-chain monoterpenoid present in the essential oils of several medicinal plants. The aim of this work was to evaluate the effects of orally administered citral in experimental models of acute and chronic nociception, inflammation, and gastric ulcers caused by non-steroidal anti-inflammatory drugs (NSAIDs). Oral treatment with citral significantly inhibited the neurogenic and inflammatory pain responses induced by intra-plantar injection of formalin. Citral also had prophylactic and therapeutic anti-nociceptive effects against mechanical hyperalgesia in plantar incision surgery, chronic regional pain syndrome, and partial ligation of sciatic nerve models, without producing any significant motor dysfunction. In addition, citral markedly attenuated the pain response induced by intra-plantar injection of glutamate and phorbol 12-myristate 13-acetate (PMA, a protein kinase C activator), as well as by intrathecal (i.t.) injection of ionotropic and metabotropic glutamate receptor agonists (N-methyl-d-aspartic acid [NMDA] and 1-amino-1,3-dicarboxycyclopentane [trans-ACPD], respectively), substance P, and cytokine tumour necrosis factor-α. However, citral potentiated behaviours indicative of pain caused by i.t., but not intra-plantar, injection of a transient receptor potential vanilloid receptor type 1 (TRPV1) agonist. Finally, the anti-nociceptive action of citral was found to involve significant activation of the 5-HT2A serotonin receptor. The effect of citral was accompanied by a gastro-protective effect against NSAID-induced ulcers. Together, these results show the potential of citral as a new drug for the treatment of pain.

Increased response to glutamate in small diameter dorsal root ganglion neurons after sciatic nerve injury.

Glutamate in the peripheral nervous system is involved in neuropathic pain, yet we know little how nerve injury alters responses to this neurotransmitter in primary sensory neurons. We recorded neuronal responses from the ex-vivo preparations of the dorsal root ganglia (DRG) one week following a chronic constriction injury (CCI) of the sciatic nerve in adult rats. We found that small diameter DRG neurons (<30 µm) exhibited increased excitability that was associated with decreased membrane threshold and rheobase, whereas responses in large diameter neurons (>30 µm) were unaffected. Puff application of either glutamate, or the selective ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA), or the group I metabotropic receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG), induced larger inward currents in CCI DRGs compared to those from uninjured rats. N-methyl-D-aspartate (NMDA)-induced currents were unchanged. In addition to larger inward currents following CCI, a greater number of neurons responded to glutamate, AMPA, NMDA, and DHPG, but not to KA. Western blot analysis of the DRGs revealed that CCI resulted in a 35% increase in GluA1 and a 60% decrease in GluA2, the AMPA receptor subunits, compared to uninjured controls. mGluR1 receptor expression increased by 60% in the membrane fraction, whereas mGluR5 receptor subunit expression remained unchanged after CCI. These results show that following nerve injury, small diameter DRG neurons, many of which are nociceptive, have increased excitability and an increased response to glutamate that is associated with changes in receptor expression at the neuronal membrane. Our findings provide further evidence that glutamatergic transmission in the periphery plays a role in nociception.

Fluorescent imaging reports an extracellular alkalinization induced by glutamatergic activation of isolated retinal horizontal cells

Extracellular acidification induced by retinal horizontal cells has been hypothesized to underlie lateral feedback inhibition onto vertebrate photoreceptors. To test this hypothesis, the H(+)-sensitive fluorophore 5-hexadecanoylaminofluorescein (HAF) was used to measure changes in H(+) from horizontal cells isolated from the retina of the catfish. HAF staining conditions were modified to minimize intracellular accumulation of HAF and maximize membrane-associated staining, and ratiometric fluorescent imaging of cells displaying primarily membrane-associated HAF fluorescence was conducted. Challenge of such HAF-labeled cells with glutamate or the ionotropic glutamate receptor agonist kainate produced an increase in the fluorescence ratio, consistent with an alkalinization response of +0.12 pH units and +0.23 pH units, respectively. This alkalinization was blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the L-type calcium channel blocker nifedipine, and lanthanum. The alkalinization reported by HAF was consistent with extracellular alkalinizations detected in previous studies using self-referencing H(+)-selective microelectrodes. The spatial distribution of the kainate-induced changes in extracellular H(+) was also examined. An overall global alkalinization around the cell was observed, with no obvious signs of discrete centers of acidification. Taken together, these data argue against the hypothesis that glutamatergic-induced efflux of protons from horizontal cells mediates lateral feedback inhibition in the outer retina.

Synthesis, receptor binding and activity of iso and azakainoids

Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction of the 1-pyrazoline NN bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting 2-pyrazoline obtained after desilylation may be reduced with NaCNBH3 to provide the trans azakainate analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described. Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid demonstrates that it is an ionotropic glutamate receptor agonist.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells

BackgroundThe bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells.MethodsDihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration.ResultsExposure of Hon and Mag (1–10 μM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 μM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production.ConclusionThis study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.