Kainate Receptor Antagonist Research Articles

Structure−Activity Relationship Studies on N3-Substituted Willardiine Derivatives Acting as AMPA or Kainate Receptor Antagonists

N3-substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU(K5)-containing kainate receptors (K(D) 0.105 +/- 0.007 microM vs kainate on native GLU(K5); K(D) 71.4 +/- 8.3 microM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant human GLU(K5), GLU(K5)/GLU(K6), and GLU(K5)/GLU(K2), K(B) values of 0.12 +/- 0.03, 0.12 +/- 0.01, and 0.18 +/- 0.02 microM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU(K6) or GLU(K7) kainate receptors or homomeric GLU(A1-4) AMPA receptors (IC(50) values > 100 microM). Thus, 43 is a potent and selective GLU(K5) receptor antagonist.

Crystal Structures of the Kainate Receptor GluR5 Ligand Binding Core Dimer with Novel GluR5-Selective Antagonists

Glutamate receptor (GluR) ion channels mediate fast synaptic transmission in the mammalian CNS. Numerous crystallographic studies, the majority on the GluR2-subtype AMPA receptor, have revealed the structural basis for binding of subtype-specific agonists. In contrast, because there are far fewer antagonist-bound structures, the mechanisms for antagonist binding are much less well understood, particularly for kainate receptors that exist as multiple subtypes with a distinct biology encoded by the GluR5-7, KA1, and KA2 genes. We describe here high-resolution crystal structures for the GluR5 ligand-binding core complex with UBP302 and UBP310, novel GluR5-selective antagonists. The crystal structures reveal the structural basis for the high selectivity for GluR5 observed in radiolabel displacement assays for the isolated ligand binding cores of the GluR2, GluR5, and GluR6 subunits and during inhibition of glutamate-activated currents in studies on full-length ion channels. The antagonists bind via a novel mechanism and do not form direct contacts with the E723 side chain as occurs in all previously solved AMPA and kainate receptor agonist and antagonist complexes. This results from a hyperextension of the ligand binding core compared with previously solved structures. As a result, in dimer assemblies, there is a 22 A extension of the ion channel linkers in the transition from antagonist- to glutamate-bound forms. This large conformational change is substantially different from that described for AMPA receptors, was not possible to predict from previous work, and suggests that glutamate receptors are capable of much larger movements than previously thought.

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.

Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3 H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: Involvement of a metabotropic glutamate receptor

We present data on the antiepileptic potency of 2-methyl-4-oxo-3 H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200–500 μM), but not α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg 2+]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 μM) decreased Glu-induced [ 35S]guanosine 5′- O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 μM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca 2+ ion transients induced by the mGluR agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1 mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.

Long-Term Depression of Kainate Receptor-Mediated Synaptic Transmission

Kainate receptors (KARs) have been shown to be involved in hippocampal mossy fiber long-term potentiation (LTP); however, it is not known if KARs are involved in the induction or expression of long-term depression (LTD), the other major form of long-term synaptic plasticity. Here we describe LTD of KAR-mediated synaptic transmission (EPSC(KA) LTD) in perirhinal cortex layer II/III neurons that is distinct from LTD of AMPAR-mediated transmission, which also coexists at the same synapses. Induction of EPSC(KA) LTD requires a rise in postsynaptic Ca(2+) but is independent of NMDARs or T-type voltage-gated Ca(2+) channels; however, it requires synaptic activation of inwardly rectifying KARs and release of Ca(2+) from stores. The synaptic KARs are regulated by tonically activated mGluR5, and expression of EPSC(KA) LTD occurs via a mechanism involving mGluR5, PKC, and PICK1 PDZ domain interactions. Thus, we describe the induction and expression mechanism of a form of synaptic plasticity, EPSC(KA) LTD.

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

Synthesis and Pharmacology of Willardiine Derivatives Acting as Antagonists of Kainate Receptors

The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord. The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLUK5.

Pharmacological characterization of stress-induced hyperthermia in DBA/2 mice using metabotropic and ionotropic glutamate receptor ligands

Accumulating evidence suggests that drugs acting on the glutamatergic system may represent promising novel therapeutic targets for the treatment of anxiety disorders. The stress-induced hyperthermia paradigm has been used widely to model some of the physiological symptoms associated with anxiety disorders and has produced results that are predictive of clinical efficacy. We have modified this paradigm to measure the autonomic consequences of stress induced by the fear of predation in mice. To evaluate the efficacy of several classes of metabotropic and ionotropic glutamate receptor ligands, as well as known anxiolytics and psychotropic comparators, in attenuating predatory-stress-induced hyperthermia. Male DBA/2 mice were implanted with radiotelemetric transmitters in the peritoneal cavity to measure stress-related increases in core body temperature, following placement in a novel cage containing soiled rat shavings. Clinically active compounds such as chlordiazepoxide (5-10 mg/kg), alprazolam (0.3-3 mg/kg), and buspirone (10-30 mg/kg) exhibited an anxiolytic profile. Assessment of glutamatergic agents indicated that the mGlu1 receptor antagonist LY456236 (10-30 mg/kg), mGlu5 receptor antagonist MPEP (10-30 mg/kg), mGlu2/3 receptor agonist LY354740 (3-10 mg/kg), mGlu2 receptor potentiator LY566332 (30 and 100 mg/kg), mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine (30-60 mg/kg), competitive NMDA receptor antagonist LY235959 (1 mg/kg), AMPA receptor antagonist GYKI-52466 (10-20 mg/kg), and glycine transporter-1 (GlyT-1) inhibitor ALX-5407 (3-10 mg/kg) dose-dependently attenuated stress-induced hyperthermia. The AMPA receptor potentiator LY451646, iGlu5 kainate receptor antagonist LY382884, glycine(B) receptor partial agonist D: -cycloserine, and GlyT-1 inhibitor ORG-24461 were ineffective in this model. Select metabotropic and ionotropic glutamate receptor ligands exhibited an anxiolytic profile, as measured by the attenuation of stress-induced hyperthermia, and may represent viable targets for the development of pharmacological treatments for anxiety-related disorders.

Two Prodrugs of Potent and Selective GluR5 Kainate Receptor Antagonists Actives in Three Animal Models of Pain

Amino acids 5 and 7, two potent and selective competitive GluR5 KA receptor antagonists, exhibited high GluR5 receptor affinity over other glutamate receptors. Their ester prodrugs 6 and 8 were orally active in three models of pain: reversal of formalin-induced paw licking, carrageenan-induced thermal hyperalgesia, and capsaicin-induced mechanical hyperalgesia.

Excitotoxic oligodendrocyte death and axonal damage induced by glutamate transporter inhibition

Glutamate uptake is crucial to terminate glutamate signaling and to prevent excitotoxicity. The present study describes the expression of functional glutamate transporters GLAST and GLT-1 in oligodendrocytes by means of electrophysiology, uptake assays, and immunocytochemistry. Inhibition of glutamate uptake, both in oligodendrocyte cultures and in isolated optic nerves, increases glutamate levels and causes oligodendrocyte excitotoxicity, which is prevented by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor antagonists. Furthermore, glutamate transporter inhibitors or antisense oligonucleotides applied onto the optic nerve in vivo lead to oligodendroglial loss, massive demyelination, and severe axonal damage. Overall, these results demonstrate that the integrity of oligodendrocytes and white matter depends on proper glutamate transporter function. Deregulated transporter activity may contribute to acute and chronic white matter damage.

Properties of external plexiform layer interneurons in mouse olfactory bulb slices

In the external plexiform layer (EPL) of the main olfactory bulb, apical dendrites of inhibitory granule cells form large numbers of synapses with mitral and tufted (M/T) cells, which regulate the spread of activity along the M/T cell dendrites. The EPL also contains intrinsic interneurons, the functions of which are unknown. In the present study, recordings were obtained from cell bodies in the EPL of mouse olfactory bulb slices. Biocytin-filling confirmed that the recorded cells included interneurons, tufted cells, and astrocytes. The interneurons had fine, varicose dendrites, and those located superficially bridged the EPL space below several adjacent glomeruli. Interneuron activity was characterized by high frequency spontaneous excitatory postsynaptic potential/currents that were blocked by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and largely eliminated by the voltage-sensitive Na + channel blocker, tetrodotoxin. Interneuron activity differed markedly from that of tufted cells, which usually exhibited spontaneous action potential bursts. The interneurons produced few action potentials spontaneously, but often produced them in response to depolarization and/or olfactory nerve (ON) stimulation. The responses to depolarization resembled responses of late- and fast-spiking interneurons found in other cortical regions. The latency and variability of the ON-evoked responses were indicative of polysynaptic input. Interneurons expressing green fluorescent protein under control of the mouse glutamic acid decarboxylase 65 promoter exhibited identical properties, providing evidence that the EPL interneurons are GABAergic. Together, these results suggest that EPL interneurons are excited by M/T cells via AMPA/kainate receptors and may in turn inhibit M/T cells within spatial domains that are topographically related to several adjacent glomeruli.

The effect of a kainate GluR5 receptor antagonist on responses of spinothalamic tract neurons in a model of peripheral neuropathy in primates

The responses of antidromically identified spinothalamic tract (STT) neurons to mechanical and thermal stimuli were compared in anesthetized normal and neuropathic monkeys before and after administration of a GluR5 kainate receptor antagonist (LY382884) into the spinal cord dorsal horn through a microdialysis fiber. Peripheral neuropathy was induced by tight ligation of the L7 spinal nerve 13–15 days prior to the experiment. STT neurons recorded in the animals with neuropathy showed increased responsiveness to weak mechanical stimuli and to heating and cooling of the skin compared to STT cells in normal animals. In both normal and the neuropathic monkeys the responses of the STT neurons to mechanical and thermal stimuli were attenuated by LY382884 application in a concentration-dependent manner. Intraspinal application of LY382884 in the neuropathic animals led to a potent reduction of those responses of the STT neurons that were aggravated by the peripheral neuropathy (weak mechanical, heat and innocuous cooling stimuli). These results suggest that kainate receptors are involved in synaptic activation of STT cells in the normal state and may also play an important role in pathological pain states such as peripheral neuropathy in primates. Kainate receptor antagonists could thus be useful for the treatment of certain forms of allodynia and hyperalgesia.

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist

Willardiine derivatives with an N 3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLU K5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLU K5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLU K5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [ 3H]kainate binding with IC 50 values >100 μM at rat GLU K6, GLU K2 or GLU K6/GLU K2. An explanation of the GLU K5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLU K5 and GLU K6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca 2+. These data provide further evidence for a role for GLU K5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLU K5-containing kainate receptors so far described.

Kainate receptor agonists and antagonists mediate tolerance to kainic acid and reduce high-affinity GTPase activity in young, but not aged, rat hippocampus.

Domoic acid acts at both kainic acid (KA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors and induces tolerance against subsequent domoic acid insult in young but not aged rat hippocampus. To determine the receptor specificity of this effect, tolerance induction was examined in hippocampal slices from young and aged rats. Slices were preconditioned by exposure to low-dose KA to activate kainate receptors, or the AMPA-receptor selective agonist (S)-5-fluorowillardiine (FW), and following washout, tolerance induction was assessed by administration of high concentrations of KA or FW (respectively). FW preconditioning failed to induce tolerance to subsequent FW challenges, while KA-preconditioned slices were significantly resistant to the effects of high-dose KA. KA preconditioning failed to induce tolerance in aged CA1. Given the lasting nature of the tolerance effect, we examined G-protein-coupled receptor function. A number of ionotropic KA receptor agonists and antagonists significantly reduced constitutive GTPase activity in hippocampal membranes from young but not aged rats. Furthermore, in young CA1, low concentrations of the AMPA/KA blocker GYKI-52466 also induced tolerance to high-dose KA. Our findings suggest that tolerance is triggered by a selective reduction in constitutive KA-sensitive G-protein activity, and that this potential neuroprotective mechanism is lost with age.

Glutamate receptor-mediated oligodendrocyte toxicity in periventricular leukomalacia: a protective role for topiramate.

Periventricular leukomalacia is a form of hypoxic-ischemic cerebral white matter injury seen most commonly in premature infants and is the major antecedent of cerebral palsy. Glutamate receptor-mediated excitotoxicity is a predominant mechanism of hypoxic-ischemic injury to developing cerebral white matter. We have demonstrated previously the protective effect of AMPA-kainate-type glutamate receptor blockade in a rodent model of periventricular leukomalacia. The present study explores the therapeutic potential of glutamate receptor blockade for hypoxic-ischemic white matter injury. We demonstrate that AMPA receptors are expressed on developing human oligodendrocytes that populate fetal white matter at 23-32 weeks gestation, the period of highest risk for periventricular leukomalacia. We show that the clinically available anticonvulsant topiramate, when administered post-insult in vivo, is protective against selective hypoxic-ischemic white matter injury and decreases the subsequent neuromotor deficits. We further demonstrate that topiramate attenuates AMPA-kainate receptor-mediated cell death and calcium influx, as well as kainate-evoked currents in developing oligodendrocytes, similar to the AMPA-kainate receptor antagonist 6-nitro-7-sulfamoylbenzo-(f)quinoxaline-2,3-dione (NBQX). Notably, protective doses of NBQX and topiramate do not affect normal maturation and proliferation of oligodendrocytes either in vivo or in vitro. Taken together, these results suggest that AMPA-kainate receptor blockade may have potential for translation as a therapeutic strategy for periventricular leukomalacia and that the mechanism of protective efficacy of topiramate is caused at least in part by attenuation of excitotoxic injury to premyelinating oligodendrocytes in developing white matter.

Astrocyte-mediated activation of neuronal kainate receptors.

Exogenous kainate receptor agonists have been shown to modulate inhibitory synaptic transmission in the hippocampus, but the pathways involved in physiological activation of the receptors remain largely unknown. Accumulating evidence indicates that astrocytes can release glutamate in a Ca(2+)-dependent manner and signal to neighboring neurons. We tested the hypothesis that astrocyte-derived glutamate activates kainate receptors on hippocampal interneurons. We report here that elevation of intracellular Ca(2+) in astrocytes, induced by uncaging Ca(2+), o-nitrophenyl-EGTA, increased action potential-driven spontaneous inhibitory postsynaptic currents in nearby interneurons in rat hippocampal slices. This effect was blocked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptor antagonists, but not by selective AMPA receptor or N-methyl-d-aspartate receptor antagonists. This pharmacological profile indicates that kainate receptors were activated during Ca(2+) elevation in astrocytes. Kainate receptors containing the GluR5 subunit seemed to mediate the observed effect because a selective GluR5-containing kainate receptor antagonist blocked the changes in sIPSCs induced by Ca(2+) uncaging, and bath application of a selective GluR5-containing receptor agonist robustly potentiated sIPSCs. When tetrodotoxin was included to block action potentials, Ca(2+) uncaging induced a small decrease in the frequency of miniature inhibitory postsynaptic currents, which was not affected by AMPA/kainate receptor antagonists. Our data suggest that an astrocyte-derived, nonsynaptic source of glutamate represents a signaling pathway that can activate neuronal kainate receptors. By modulating the activity of interneurons, astrocytes may play a critical role in circuit function of hippocampus.

Local γ‐aminobutyric acid and glutamate circuit control of hypophyseotrophic corticotropin‐releasing factor neuron activity in the paraventricular nucleus of the hypothalamus

Paraventricular corticotropin-releasing factor (CRF) neurons play a pivotal role in regulating neuroendocrine responses to stress. The mechanisms by which synaptic inputs control the activity of these neurons are not well understood. The present study was undertaken to determine the role of the intrinsic gamma-aminobutyric acid (GABA)- and glutamatergic neural circuits of the hypothalamic paraventricular nucleus (PVN) in the control of CRF neural activity. We show that in organotypic cultures of the PVN, blockade of the intrinsic GABAergic neurotransmission by the GABAA receptor antagonist bicuculline resulted in a significant increase in CRF secretion. The bicuculline-induced CRF secretory activity was abolished by the coadministration of the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Electrical stimulation of the CRF cell division elicited glutamatergic extracellular field potentials that were dramatically enhanced by bicuculline and were suppressed by CNQX. These results show that the functional activity of CRF neurons in organotypic cultures of the PVN is under a tonic inhibitory influence of an intrinsic GABAergic circuit. Suppression of GABAergic transmission appears to have a permissive role for inducing an increased secretory activity of CRF neurons that is driven by an excitatory glutamatergic network via AMPA/kainate receptors.

CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell–collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1− to BrdU−/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-μM bicuculline, and completely blocked by additional 10 μM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.

Glutamate transporters regulate excitability in local networks in rat neocortex

Excitatory postsynaptic currents (EPSCs) in the neocortex are principally mediated by glutamate receptors. Termination of excitation requires rapid removal of glutamate from the synaptic cleft following release. Glutamate transporters are involved in EPSC termination but the effect of uptake inhibition on excitatory neurotransmission varies by brain region. Epileptiform activity is largely mediated by a synchronous synaptic activation of cells in local cortical circuits, presumably associated with a large release of glutamate. The role of glutamate transporters in regulating epileptiform activity has not been addressed. Here we examine the effect of glutamate transport inhibition on EPSCs and epileptiform events in layer II/III pyramidal cells in rat neocortex. Inhibiting glutamate transporters with dl-threo-β-benzyloxyaspartic acid (TBOA; 30 μM) had no effect on the amplitude or decay time of evoked, presumably α-amino-3-hydroxyl-5-methyl-isoxazolepropionic acid-mediated, EPSCs. In contrast, the amplitude and duration of epileptiform discharges were significantly enhanced. TBOA resulted also in a decreased threshold for evoking epileptiform activity and an increased probability of occurrence of spontaneous epileptiform discharges. TBOA's effects were not inhibited by the group I and II metabotropic glutamate receptors antagonist ( S)-α-methyl-4-carboxyphenylglycine or the kainate receptor antagonist [(3 S,4a R, 6 S, 8a R)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid]. d-(−)-2-amino-5-phosphonovaleric acid could both prevent excitability changes by TBOA and block already induced changes. Dihydrokainate (300 μM) had effects similar to TBOA suggesting involvement of the glial transporter GLT-1. Inhibiting glutamate transport increases local network excitability under conditions where there is an enhanced release of glutamate. Our results indicate that uptake inhibition produces an elevation of extracellular glutamate levels and activation of N-methyl- d-aspartate receptors.