NT-3 evokes an LTP-like facilitation of AMPA/kainate receptor-mediated synaptic transmission in the neonatal rat spinal cord

Neurotrophin-3 (NT-3) is a neurotrophic factor required for survival of muscle spindle afferents during prenatal development. It also acts postsynaptically to enhance the monosynaptic excitatory postsynaptic potential (EPSP) produced by these fibers in motoneurons when applied over a period of weeks to the axotomized muscle nerve in adult cats. Similar increases in the amplitude of the monosynaptic EPSP in motoneurons are observed after periodic systemic treatment of neonatal rats with NT-3. Here we show an acute action of NT-3 in enhancing the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptor-mediated fast monosynaptic EPSP elicited in motoneurons by dorsal root (DR) stimulation in the in vitro hemisected neonatal rat spinal cord. The receptor tyrosine kinase inhibitor K252a blocks this action of NT-3 as does the calcium chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) injected into the motoneuron. The effect of NT-3 resembles long-term potentiation (LTP) in that transient bath application of NT-3 to the isolated spinal cord produces a long-lasting increase in the amplitude of the monosynaptic EPSP. An additional similarity is that activation of N-methyl-D-aspartate (NMDA) receptors is required to initiate this increase but not to maintain it. The NMDA receptor blocker MK-801, introduced into the motoneuron through the recording microelectrode, blocks the effect of NT-3, indicating that NMDA receptors in the motoneuron membrane are crucial. The effect of NT-3 on motoneuron NMDA receptors is demonstrated by its enhancement of the depolarizing response of the motoneuron to bath-applied NMDA in the presence of tetrodotoxin (TTX). The potentiating effects of NT-3 do not persist beyond the first postnatal week. In addition, EPSPs with similar properties evoked in the same motoneurons by stimulation of descending fibers in the ventrolateral funiculus (VLF) are not modifiable by NT-3 even in the initial postnatal week. Thus, NT-3 produces synapse-specific and age-dependent LTP-like enhancement of AMPA/kainate receptor-mediated synaptic transmission in the spinal cord, and this action requires the availability of functional NMDA receptors in the motoneuron.     

Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.     

Corticostriatal paired-pulse potentiation produced by voltage-dependent activation of NMDA receptors and L-type Ca(2+) channels.

AMPA and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses expressed differential paired-pulse plasticity when examined in the same cell using intracellular or whole cell voltage-clamp recordings. Electrical stimulation of corticostriatal afferents in brain slices bathed in artificial cerebrospinal fluid containing bicuculline produces excitatory postsynaptic potentials and excitatory postsynaptic currents (EPSCs) mediated primarily by AMPA receptors. Cell-to-cell variation existed in AMPA receptor paired-pulse plasticity, but within-cell plasticity was stable over a range of stimulation intensities. Addition of 6-cyano-7-nitroquinoxalene-2,3-dione blocked most of the synaptic response leaving behind a small AP-5-sensitive component. Increasing the stimulation intensity produced large, long-lasting NMDA receptor-mediated responses. In contrast to AMPA receptor-mediated responses, NMDA receptor responses consistently showed an increase in paired-pulse potentiation with increasing stimulation intensity. This relationship was restricted to interstimulus intervals shorter than 100 ms. Paired-pulse potentiation of NMDA receptor responses was voltage-dependent and reduced by removal of extracellular Mg(2+). Block of postsynaptic L-type Ca(2+) channels with nifedipine produced a voltage-dependent reduction of NMDA receptor excitatory postsynaptic currents (EPSCs) and a voltage-dependent reduction of NMDA receptor paired-pulse potentiation. These data indicate depolarization during the first NMDA receptor response causes facilitation of the second by removing voltage-dependent block of NMDA receptors by Mg(2+) and by activating voltage-dependent Ca(2+) channels.     

Differential NR2B subunit expression at dorsal root and ventrolateral funiculus synapses on lumbar motoneurons of neonatal rat

Synapse specific differences in NR2 subunit expression exist in several systems within the mammalian CNS. Here we have studied such differences on motoneurons in the neonatal rat cord using ifenprodil known to inhibit voltage-, use- and glycine-independent responses mediated by NR2B-containing N-methyl-d-aspartate receptors (NMDARs) with high specificity. In neonatal rats (P1–P9), the synapses made by the dorsal root (DR) fibres were more sensitive to ifenprodil than ventrolateral funiculus (VLF) connections on the same motoneuron. DR connections exhibited very little additional blockade to bath-applied MK-801 whereas VLF connections displayed a further decrease in amplitude. This suggests that at this immediate postnatal age, DR synapses on motoneurons contain a higher proportion of ifenprodil-sensitive diheteromeric NR1/NR2B receptors than VLF synapses. Since DR synapses have been shown in other studies to be less mature than VLF synapses on the same motoneuron at this developmental stage, these data are interpreted as indicating that less mature NMDA receptors feature a higher proportion of NR2B subunits which declines as the synapse matures. This novel finding of staggered development of NMDA receptors from different synaptic inputs on the same motoneuron is discussed in the context of its developmental and functional implications.     

AMPA receptor-mediated miniature synaptic calcium transients in GluR2 null mice

AMPA-type glutamate receptors are normally Ca(2+) impermeable due to the expression of the GluR2 receptor subunit. By using GluR2 null mice we were able to detect miniature synaptic Ca(2+) transients (MSCTs) associated with AMPA-type receptor-mediated miniature synaptic currents at single synapses in primary cortical cultures. MSCTs and associated Ca(2+) transients were monitored under conditions that isolated responses mediated by AMPA or N-methyl-D-aspartate (NMDA) receptors. As expected, addition of the antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 3 microM) blocked the AMPA receptor-mediated MSCTs. Voltage-gated Ca(2+) channels did not contribute to AMPA MSCTs because CdCl(2) (0.1-0.2 mM) did not significantly alter the frequency or the amplitude of the MSCTs. The amplitude of AMPA MSCTs appeared to be regulated independently from event frequency since the two measures were not correlated (R = 0.023). Synapses were identified that only expressed MSCTs attributed to either NMDA or AMPA receptors. At synapses with only NMDA responses, MSCT amplitude was significantly lower (by 40%) than synapses expressing both NMDA and AMPA responses. At synapses that showed MSCTs mediated by both AMPA and NMDA receptors, the amplitude of the transients in each condition was positively correlated (R = 0.94). Our results suggest that when AMPA and NMDA receptors are co-expressed at synapses, mechanisms exist to ensure proportional scaling of each receptor type that are distinct from the presynaptic factors controlling the frequency of miniature release.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Modulation of NMDA receptor properties and synaptic transmission by the NR3A subunit in mouse hippocampal and cerebrocortical neurons

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-d-aspartate (NMDA)-induced currents and decreased Mg(2+) sensitivity and Ca(2+) permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg(2+) sensitivity, and decreased Ca(2+) permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg(2+) sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extrasynaptic receptors, likely composed of NR1, NR2, and NR3 subunits.     

Light-evoked excitatory synaptic currents of X-type retinal ganglion cells

The excitatory amino acid receptor (EAAR) types involved in the generation of light-evoked excitatory postsynaptic currents (EPSCs) were examined in X-type retinal ganglion cells. Using isolated and sliced preparations of cat and ferret retina, the light-evoked EPSCs of X cells were isolated by adding picrotoxin and strychnine to the bath to remove synaptic inhibition. N-methyl-D-aspartate (NMDA) receptors contribute significantly to the light-evoked EPSCs of ON- and OFF-X cells at many different holding potentials. An NMDA receptor contribution to the EPSCs was observable when retinal synaptic inhibition was either normally present or pharmacologically blocked. NMDA receptors formed 80% of the peak light-evoked EPSC at a holding potential of -40 mV; however, even at -80 mV, 20% of the light-evoked EPSC was NMDA-mediated. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-mediated component to the light-evoked EPSCs predominated at a holding potential of -80 mV. The light-evoked EPSC was blocked by the AMPA receptor-selective antagonist GYKI52466 (50-100 microM). The AMPA receptor-mediated EPSC component had a linear current-voltage relation. AMPA receptors form the main non-NMDA EAAR current on both ON- and OFF- X ganglion cell dendrites. When synaptic transmission was blocked by the addition of Cd(2+) to the Ringer, application of kainate directly to ganglion cells evoked excitatory currents that were strongly blocked by GYKI52466. Experiments using selective EAAR modulators showed the AMPA receptor-selective modulator cyclothiazide potentiated glutamate-evoked currents on X cells, while the kainate receptor-selective modulator concanavalin A (ConA) had no effect on kainate-evoked currents. Whereas the present study confirms the general notion that AMPA EAAR-mediated currents are transient and NMDA receptor-mediated currents are sustained, current-voltage relations of the light-evoked EPSC at different time points showed the contributions of these two receptor types significantly overlap. Both NMDA and AMPA EAARs can transmit transient and sustained visual signals in X ganglion cells, suggesting that much signal shaping occurs presynaptically in bipolar cells.     

Metabotropic glutamate receptors modify ionotropic glutamate responses in neocortical pyramidal cells and interneurons

In neocortex glutamate activates ionotropic and metabotropic receptors (mGluRs). Whole-cell current-clamp recordings in the in vitro rat auditory cortex at 32 degrees C were used to explore the role that mGluRs have in regulation of AMPA/kainate, NMDA, and GABA receptor-mediated synaptic transmission. Our findings are: (a) The fast EPSP (AMPA/kainate), slow EPSP (NMDA), and IPSPs (GABAA, GABAB), elicited in pyramidal neurons are reduced in the presence of (1S,3R)-ACPD (mGluR agonist) with greatest effect on the slow IPSP>fast IPSP>fast EPSP. The effect is likely the result of ACPD acting at presynaptic mGluRs because the probability of release of glutamate and GABA is reduced in the presence of ACPD, intracellular infusion of a G protein antagonist (GDPPS) did not block the effect of ACPD, nor were iontophoretic kainic acid or NMDA-induced depolarizations reduced by ACPD. (b) The slow EPSP is enhanced following washout of ACPD and enhancement is not due to disinhibition because it is present in the absence of IPSPs, but if IPSPs are present, its magnitude can be influenced. Iontophoretic NMDA responses are enhanced in the presence of ACPD, an effect blocked by GDPbetaS and heparin (intracellular inositol 1,4,5-trisphosphate receptor antagonist). Taken together, this evidence suggests that enhancement is a result of group I postsynaptic mGluR activation. (c) In fast-spiking cells ACPD reduces the EPSP (AMPA/kainate and NMDA-mediated). This action is likely presynaptic because it persists when GDPbetaS is in the cells. (d) The rate of spike discharge recorded from fast-spiking cells is accelerated in ACPD but does not change in the presence of GDPbetaS, suggesting a postsynaptic effect. Our data indicate that mGluRs can influence neocortical synaptic transmission in complex ways by acting presynaptically and postsynaptically.     

Properties of miniature glutamatergic EPSCs in neurons of the locomotor regions of the developing zebrafish

As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg(2+), a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.     

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons.The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.     

Pattern generation in caudal-lumbar and sacrococcygeal segments of the neonatal rat spinal cord

Glutamate is the major excitatory amino acid neurotransmitter in the CNS, including the neocortex, hippocampus, and spinal cord. Normal synaptic transmission is mainly mediated by glutamate AMPA and/or kainate receptors. Glutamate N-methyl-D-aspartate (NMDA) receptors are normally inactive and only activated when a sufficient postsynaptic depolarization is induced by the activity. Here we show that in sensory synapses of adult mouse, some synaptic responses (26.3% of a total of 38 experiments) between primary afferent fibers and dorsal horn neurons are almost completely mediated by NMDA receptors. Dorsal root stimulation did not elicit any detectable AMPA/kainate receptor-mediated responses in these synapses. Unlike young spinal cord, serotonin alone did not produce any long-lasting synaptic enhancement in adult spinal dorsal horn neurons. However, co-application of the adenylyl cyclase activator forskolin and serotonin (5-HT) produced long-lasting enhancement, including the recruitment of functional AMPA receptor-mediated responses. Calcium-sensitive, calmodulin-regulated adenylyl cyclases (AC1, AC8) are required for the enhancement. Furthermore the thresholds for generating action potential responses were decreased, and, in many cases, co-application of forskolin and 5-HT led to the generation of action potentials by previously subthreshold stimulation of primary afferent fibers in the presence of the NMDA receptor blocker 2-amino-5-phosphonovaleric acid. Our results suggest that pure NMDA synapses exist on sensory neurons in adult spinal cord and that they may contribute to functional sensory transmission. The synergistic recruitment of functional AMPA responses by 5-HT and forskolin provides a new cellular mechanism for glutamatergic synapses in mammalian spinal cord.     

Different composition of glutamate receptors in corticothalamic and lemniscal synaptic responses and their roles in the firing responses of ventrobasal thalamic neurons in juvenile mice

Thalamic ventrobasal (VB) relay neurons receive information via two major types of glutamatergic synapses, that is, from the medial lemniscus (lemniscal synapses) and primary somatosensory cortex (corticothalamic synapses). These two synapses influence and coordinate firing responses of VB neurons, but their precise operational mechanisms are not yet well understood. In this study, we compared the composition of glutamate receptors and synaptic properties of corticothalamic and lemniscal synapses. We found that the relative contribution of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to non-NMDA receptor-mediated EPSCs was significantly greater in corticothalamic synapses than in lemniscal synapses. Furthermore, NMDA receptor 2B-containing NMDA receptor- and kainate receptor-mediated currents were observed only in corticothalamic synapses, but not in lemniscal synapses. EPSCs in corticothalamic synapses displayed the postsynaptic summation in a frequency-dependent manner, in which the summation of the NMDA receptor-mediated component was largely involved. The summation of kainate receptor-mediated currents also partially contributed to the postsynaptic summation in corticothalamic synapses. In contrast, the contribution of NMDA receptor-mediated currents to the postsynaptic summation of lemniscal EPSCs was relatively minor. Furthermore, our results indicated that the prominent NMDA receptor-mediated component in corticothalamic synapses was the key determinant for the late-persistent firing of VB neurons in response to corticothalamic stimuli. In lemniscal synapses, in contrast, the onset-transient firing in response to lemniscal stimuli was regulated mainly by AMPA receptors.     

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes)

Ionotropic glutamate receptor agonists, kainate, -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl- -aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release.

We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.     

Kainate receptors at corticothalamic synapses do not contribute to synaptic responses

Kainate receptors (KAR) remain the most poorly defined components of the glutamate receptor system in the CNS, mainly because of the difficulty of distinguishing currents gated by KAR from those mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor activation, and because KAR are expressed at significantly lower levels than AMPA receptors in most parts of the CNS.The corticothalamic projection exerts its effects on thalamic neurons via NMDA, non-NMDA and metabotropic glutamate receptors. AMPA receptor mediated effects tend to predominate in the mature thalamus, but the involvement of kainate receptors at corticothalamic synapses on relay neurons and reticular nucleus neurons had not been studied.The present work compared KAR influences on neurons in the ventral posterior nucleus (VP) and reticular nucleus (RTN), using whole-cell recording in P14–P20 mouse thalamocortical slices. The results were correlated with quantitative immuno-electron microscopic localization of kainate receptor sub-units at corticothalamic synapses in these nuclei. Small kainate-induced inward currents could be recorded in thalamic neurons in response to bath application of kainate, but no KAR-mediated pre-synaptic effects could be detected and no synaptic responses could be evoked in these cells by corticothalamic stimulation. Morphologically, GluR5/6/7 sub-units were expressed at low levels in both VP and RTN and were confined to post-synaptic membranes at corticothalamic synapses in both VP and RTN. Many synapses, however, lacked GluR5/6/7 immunoreactivity.These results suggest that kainate receptor-mediated events are not major components of the responses of thalamic neurons to corticothalamic activation, either because of small numbers or their location in sites inaccessible to glutamate released from corticothalamic terminals.     

Functional roles of protein interactions with AMPA and kainate receptors

The glutamate receptor subtypes AMPA and kainate are involved in synaptic transmission and synaptic plasticity in the CNS. Recently there has been considerable interest in understanding the molecular regulation of these receptors by proteins that directly bind to AMPA and kainate receptor subunits. Amongst the first interaction partners to be discovered were NSF, ABP, GRIP and PICK1, which bind the AMPA receptor subunit GLUA2. We have studied the functional roles of the interactions of these proteins in regulating AMPA receptor-mediated synaptic transmission and synaptic plasticity in the hippocampus. We have also started to investigate the functions of PICK1 and GRIP on kainate receptor-mediated synaptic transmission in this region. In this article we reflect upon this work, which has led to some new ideas about how AMPA and kainate receptors are regulated at synapses.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats

Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.     

Serotonin type II receptor activation facilitates synaptic plasticity via N-methyl-D-aspartate-mediated mechanism in the rat basolateral amygdala

The modulation of synaptic plasticity by serotonin type II (5-hydroxytryptamine type II (5-HT(2)))-receptor stimulation was explored using intracellular, field potential and Fura-2 fluorescence image recordings in a rat amygdala slice preparation. Bath application of 5HT(2) receptor agonist 1-(2,5)-dimethoxy-4-iodophen-2-aminopropane (DOI) transformed theta-burst-stimulated (TBS) synaptic plasticity from short-term potentiation to long-term potentiation. DOI enhanced N-methyl-D-aspartate (NMDA) receptor-mediated potentials and calcium influx without affecting the resting membrane potential or input resistance of the neurons. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated excitatory synaptic responses were unaffected by DOI. The facilitating effects of DOI were blocked by the 5-HT(2) receptor antagonist, ketanserin, and by the 5-HT(2C)-receptor selective antagonist, RS102221. These results indicate that 5-HT(2)-receptor activation enhances NMDA receptor-mediated synaptic function in the basolateral amygdala (BLA).     

Impaired glutamatergic synaptic transmission in the PKU brain

This paper reviews recent results of our investigation of the mechanisms whereby hyperphenylalaninemia may cause brain dysfunction in classical phenylketonuria (PKU). Acute applications of L-Phe in rat and mouse hippocampal and cerebrocortical cultured neurons, at a range of concentrations found in PKU brain, significantly and reversibly depressed glutamatergic synaptic transmission by a combination of pre- and postsynaptic actions: (1) competition for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptors; (2) attenuation of neurotransmitter release; (3) competition for the glutamate-binding site of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid and kainate (AMPA/kainate) receptors. Unlike L-Phe, its non-tyrosine metabolites, phenylacetic acid, phenylpyruvic acid, and phenyllactic acid, did not produce antiglutamatergic effects. L-Phe did not affect inhibitory gamma-aminobutyric (GABA)-ergic transmission. Consistent with this specific pattern of effects caused by L-Phe in neuronal cultures, the expression of NMDA receptor NR2A and AMPA receptor Glu1 and Glu2/3 subunits in brain of hyperphenylalaninemic PKU mice (Pah(enu2) strain) was significantly increased, whereas expression of the NMDA receptor NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Considering the important role of glutamatergic synaptic transmission in normal brain development and function, these L-Phe-induced changes in glutamatergic synaptic transmission in PKU brain may be a critical element of the neurological symptoms of PKU.