Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells.

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.

Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation.

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

GluR2 expressed by glial fibrillary acidic protein promoter decreases the number of neurons.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) type ionotropic glutamate receptors participate in fast neuronal transmission. GluR2, a subunit of AMPA receptors, is the determinant of Ca2+-permeability and surface expression of the receptors. To elucidate the role of AMPA receptors in the cells expressing glial fibrillary acidic protein (GFAP), we constructed mice expressing rat GluR2 cDNA under the control of a human GFAP promoter. The mice expressed approximately two folds increase of GluR2 in primary culture of astrocytes. Colocalization of GluR2 and GFAP was observed in Bergmann glial cells, which normally expressed AMPA receptors lacking GluR2. The diameter of glial fibers was significantly reduced and the leading edge of the processes was thinning or retracted in primary cultured BG cells. Interestingly, the transgenic mice had smaller brains compared with wild type mice. We found a 32% decrease in the number of cerebellar granule cells and a 31% decreases in cerebral cortical neurons. These results indicate that the increased expression of GluR2 in GFAP-positive cells alters neuron-glial interaction and leads to reduction in the number of neurons in adult mice.

Postsynaptic expression of a new calcium pathway in hippocampal CA3 neurons and its influence on mossy fiber long-term potentiation.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is induced by postsynaptic Ca(2+) influx via NMDA receptors (NMDARs). However, this synaptic plasticity occurs independently of NMDARs when Ca(2+)-permeable AMPA receptors (AMPARs) are expressed at postsynaptic sites using various genetic techniques, indicating that an increase in Ca(2+) level at critical postsynaptic sites, regardless of its entry pathway, triggers the induction of LTP at CA1 synapses. In contrast, NMDARs are sparsely distributed on mossy fiber (MF) synapses in CA3 hippocampal neurons, and most evidence favors the presynaptic mechanism for LTP induction, although some reports suggested a postsynaptic mechanism. In this study, we examined whether Ca(2+) influx through the newly produced postsynaptic receptors during high-frequency stimulation affects the induction of MF LTP. For this purpose, we expressed Ca(2+)-permeable AMPARs in CA3 pyramidal neurons by Sindbis viral-mediated gene transfer of the unedited form of the glutamate receptor 2 (GluR2Q) subunit, as a new pathway for postsynaptic Ca(2+) entry, in rat hippocampal organotypic cultures. Virally expressed myc-tagged GluR2Q was detected at the complex spines known as the thorny excrescences, which serve as postsynaptic targets for MF synaptic input, on the proximal apical dendrites of CA3 pyramidal cells. Furthermore, endogenous Ca(2+)-impermeable AMPARs at MF synapses were converted into Ca(2+)-permeable receptors by GluR2Q expression. However, the postsynaptic expression of Ca(2+)-permeable AMPARs had no significant influence on the two types of MF LTP induced by different stimulus protocols. These results supported the notion that MF LTP is independent of postsynaptic Ca(2+).

Identification and characterization of a novel Delphilin variant with an alternative N-terminus.

Delphilin is identified as a Glutamate receptor delta2 (GluRdelta2) subunit interacting protein, consisting of a PDZ domain and formin homology (FH) domains 1 and 2, in addition to a C-terminal coiled-coil structure. Delphilin has been shown to be selectively expressed in cerebellar Purkinje cells where it co-localizes with the GluRdelta2 subunit at the Purkinje cell-parallel fiber synapses. Although Delphilin specifically interacts with the GluRdelta2 C-terminus via its PDZ domain, the physiological role of the interaction is not yet understood. Here, we report that the Delphilin protein exhibits diversity at its N-terminus by variable usage of the first several exons. Interestingly, the two Delphilin mRNAs which correspond to the first one initially identified (now designated as Delphilin alpha) and the second that contains a newly identified first exon (designated as Delphilin beta), show different chronological expression profiles. Delphilin beta mRNA was not decreased throughout the cerebellar development in vivo and in vitro, while in vivo Delphilin alpha mRNA gradually decreases following the first postnatal week. Delphilins alpha and beta also revealed different subcellular distribution with some overlap. Specifically, the cerebellar synaptosomal membrane fraction contained the Delphilin beta protein. Both Delphilin alpha and beta localized at the dendritic spines with GluRdelta2; however, dendritic shafts in cultured Purkinje cells also included Delphilin beta. In MDCK cells upon becoming confluent, Delphilin alpha moved to the cell-cell junction area, whereas Delphilin beta maintained a diffuse distribution pattern throughout the cytoplasm. Taken as a whole, these two different Delphilins seemed to play functionally different roles in developing and matured cerebellar Purkinje cells.

Transfer of NMDAR2 cDNAs increases endogenous NMDAR1 protein and induces expression of functional NMDA receptors in PC12 cells.

The pheochromocytoma cell line (PC12) has been used as a model system for the study of regulation of expression of NMDA receptors. PC12 cells express a substantial amount of NMDAR1 subunit (NR1) mRNA, whereas they express only a small amount of NR1 protein. The level of functional NMDA receptor expression is almost negligible. To test the possibility that NMDAR2 subunits (NR2) control expression of functional NMDA receptors as well as NR1 protein, we transferred NR2A-D cDNAs into PC12 cells using adenovirus vectors. Prominent NMDA receptor-mediated currents were recorded in PC12 cells to which NR2A or NR2B cDNA was delivered without NR1 cDNA. The amplitudes of these responses were similar to those in PC12 cells to which NR1 cDNA was delivered together with NR2A or NR2B cDNA. In cells to which either NR2C or NR2D cDNA alone was delivered, NMDA receptor-mediated currents were also detected, although to a much lesser extent. These results showed that NR2 proteins produced by gene transfer are co-assembled with the endogenous NR1 protein to form functional heteromeric receptors. The delivery of NR2A-D cDNAs also increased the amount of NR1 protein but not that of NR1 mRNA, suggesting that this protein increase is due to post-transcriptional mechanisms. The effects of NR2A-B gene transfer on expression of NR1 protein were much more efficient than those of NR2C-D gene transfer.

Overexpression of Ca2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia.

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Predominant expression of GluR2 among the AMPA receptor subunits in neuronal progenitor cells of the rat hippocampus.

Cells derived from the hippocampus of embryonic day 18 (E18) rats were cultured in B27-supplemented Neurobasal medium without serum. We found the presence of numerous small cells with round or elliptical somata and fine processes in this primary culture. These cells were first detectable on culture day 8 and gradually increased in number that reached the maximum on approximately day 14. They incorporated bromodeoxyuridine (BrdU) and expressed nestin, a marker of stem cells and progenitor cells. Furthermore, nearly a half of these cells also expressed neuron-specific beta tubulin. On the other hand, they did not express O4 and glial fibrillary acid protein (GFAP), markers of oligodendrocytes and astrocytes, respectively. Thus, these small cells are most likely to be neuronal progenitor cells. The whole-cell patch clamp studies revealed that these cells expressed voltage-gated Na+, Ca2+ and K+ channels. With regard to ligand-gated channels, these cells were sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), but not to N-methyl-D-aspartate (NMDA). The current-voltage relationship of the AMPA-induced current was slightly outwardly rectifying, suggesting that the AMPA receptors contained the GluR2 subunit in their oligomeric assemblies. The single-cell reverse transcription (RT)-PCR analysis revealed that GluR2 is predominant over the other AMPA receptor subunits in these cells. Furthermore, GluR2 was expressed mainly in the flip form.

Serum-dependence of AMPA receptor-mediated proliferation in glioma cells.

Glutamate may cause Ca(2+) entry through Ca(2+)-permeable glutamate receptors, which in turn stimulates the anti-apoptotic signaling cascade in glioma cells. It was found that a human glioma cell line, U-87 MG, expressed subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate acid-type glutamate receptors (AMPAR). Ca(2+) entry through AMPAR was detected in approximately 40% of U-87 MG cells. AMPAR agonists facilitated cell proliferation in low-serum medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell proliferation by the activation of AMPAR was not detected in serum-rich medium containing 10% FCS. Overexpression of Ca(2+)-permeable AMPAR facilitated proliferation of U-87 MG cells in the low-serum condition, whereas it had again no effect in the serum-rich condition. Cell proliferation of U-87 MG cells is likely to be under the regulation of both growth factors contained in the serum and Ca(2+) entry through AMPAR, and that the latter regulation becomes evident only when serum factors are deprived of culture medium.

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis.

Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.

Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution.

Aequorin is a photoprotein that emits light upon binding calcium. Aequorin mutants showing increased intensity or slow decay of bioluminescence were isolated by in vitro evolution combining DNA shuffling and functional screening in bacteria. Luminescence decay mutants were isolated at the first round of screening and carried mutations located in EF-hand calcium binding sites or their vicinity. During in vitro evolution, the luminescence intensity of the population of mutants increased with the frequency of effective mutations whereas the frequency of other amino acid substitutions remained roughly stable. Luminescence intensity mutations neighbored the His-16 or His-169 coelenterazine binding residues or were located in the first EF-hand. None of the selected mutants exhibited an increase in photon yield when examined in a cell-free assay. However, we observed that two mutants, Q168R and L170I, exhibited an increase of the photoprotein lifetime at 37 degrees C that may underlie their high luminescence intensity in bacteria. Further analysis of Q168R and L170I mutations showed that they increased aequorin thermostability. Conversely, examination of luminescence decay mutants revealed that the F149S substitution decreased aequorin thermostability. Finally, screening of a library of random Gln-168 and Leu-170 mutants confirmed the involvement of both positions in thermostability and indicated that optimal thermostability was conferred by Q168R and L170I mutations selected through in vitro evolution. Our results suggest that Phe-149 and Gln-168 residues participate in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues.

Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells.

The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral-mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN-EG-NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18-24 h postinfection. In most of GFP-positive cells, the expression of NR2B protein was detected by immunostaining with NR2B-specific antibodies. In Purkinje cells infected with SIN-EG-NR2B, the iontophoretic application of NMDA induced prominent NMDAR-mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR-mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.

Recent advances in the study of AMPA receptors.

As glutamate is a dominant excitatory neurotransmitter in the central nervous system, glutamate receptors, and especially AMPA receptors, are located ubiquitously in all brain areas. In this paper, we reviewed recent advances of studies on AMPA receptor functions. AMPA receptors are cation-conducting complexes composed of various combinations of four subunits (GluR1 to GluR4). The glutamine residue located in the pore-forming segment of GluR2 subunit (Q/R site) is changed to arginine by RNA editing at the pre mRNA stage in normal adult mammalian animal. The edited GluR2 subunit is a major determination of Ca(2+) permeability of the AMPA receptor; only edited GluR2-lacking receptor shows high-Ca(2+) permeability. The assembly of glutamate AMPA receptor subunit is not completely according to the stochastic theory. The heteromeric subunits assembly is more rapid than the homomeric assembly is. The transfer of AMPA receptor subunit to the plasma membrane is conducted in multiple ways. Many molecules that interact with the intracellular domain of AMPA receptor subunits are reported as the modulators of AMPA receptor subunit transfer. In the motoneuron of sporadic amyotrophic lateral sclerosis (ALS) patients, the efficiency of RNA editing at the GluR2 Q/R site is significantly decreased. Relative low level of edited GluR2 subunit expression is likely responsible for motoneuronal death in ALS. Recently, AMPA receptors in glial cells have been studied. Bergmann glial cells in cerebellum express Ca(2+)-permeable AMPA receptors. Conversion of these AMPA receptors to Ca(2+)-impermeable type receptors induces morphological and functional changes. Glioblastoma cells also express Ca(2+)-permeable AMPA receptors, and their conversion to Ca(2+)-impermeable receptors inhibits cell locomotion and induces apoptosis.

Membrane and firing properties of glutamatergic and GABAergic neurons in the rat medial vestibular nucleus.

In previous studies, neurons in the medial vestibular nucleus (MVN) were classified mainly into 2 types according to their intrinsic membrane properties in in vitro slice preparations. However, it has not been determined whether the classified neurons are excitatory or inhibitory ones. In the present study, to clarify the relationship between the chemical and electrophysiological properties of MVN neurons, we explored mRNAs of cellular markers for GABAergic (glutamic acid decarboxylase 65, 67, and neuronal GABA transporter), glutamatergic (vesicular glutamate transporter 1 and 2), glycinergic (glycine transporter 2), and cholinergic neurons (choline acetyltransferase and vesicular acetylcholine transporter) expressed in electrophysiologically characterized MVN neurons in rat brain stem slice preparations. For this purpose, we combined whole cell patch-clamp recording analysis with single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis. We examined the membrane properties such as afterhyperpolarization (AHP), firing pattern, and response to hyperpolarizing current pulse to classify MVN neurons. From the single-cell RT-PCR analysis, we found that GABAergic neurons consisted of heterogeneous populations with different membrane properties. Comparison of the membrane properties of GABAergic neurons with those of other neurons revealed that AHPs without slow components and a firing pattern with delayed spike generation (late spiking) were preferential properties of GABAergic neurons. On the other hand, most glutamatergic neurons formed a homogeneous subclass of neurons exhibiting AHPs with slow components, repetitive firings with constant interspike intervals (continuous spiking), and time-dependent inward rectification in response to hyperpolarizing current pulses. We also found a small number of cholinergic neurons with various membrane properties. These findings clarify the electrophysiological properties of excitatory and inhibitory neurons in the MVN, and the information about the preferential membrane properties may be useful for identifying GABAergic and glutamatergic MVN neurons electrophysiologically.

Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning.

NMDA receptor-dependent long-term potentiation (LTP) at hippocampal synapses has been considered a crucial component of the cellular basis for learning and memory. This form of LTP occurs in excitatory synapses in both the CA1 area and the dentate gyrus in the hippocampus. However, differential roles of LTP in these areas have not yet been identified. To address this issue, we enhanced the degree of LTP by expressing Ca2+-permeable AMPA receptors at either hippocampal CA1 or dentate gyrus synapses using Sindbis viral vectors (SINs) encoding both green fluorescent proteins and unedited GluR2 (GluR2Q) subunits, and examined their effects on rat spatial learning. The viral vectors were locally injected into the 8-week-old-rat brain in vivo bilaterally. The postsynaptic expression of Ca2+-permeable AMPA receptors enhanced the degree of LTP, and induced NMDA receptor-independent LTP in the presence of the NMDA receptor antagonist in SIN-infected regions in both CA1 and dentate gyrus in hippocampal slice preparations. However, the regional expression of Ca2+-permeable AMPA receptors caused opposite behavioural consequences on the Morris water maze task: rats with SIN-infected CA1 pyramidal cells showed shorter escape latency and better probe test performance, whereas those with SIN-infected dentate gyrus granule cells showed impaired performance. Thus, it was demonstrated that CA1 and dentate gyrus synapses play different functional roles in spatial learning despite their similar mechanism for LTP induction.

Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses.

Hippocampal CA3 pyramidal neurons receive synaptic inputs from both mossy fibres (MFs) and associational fibres (AFs). Long-term potentiation (LTP) at these synapses differs in its induction sites and N-methyl-D-aspartate receptor (NMDAR) dependence. Most evidence favours the presynaptic and postsynaptic mechanisms for induction of MF LTP and AF LTP, respectively. This implies that molecular and functional properties differ between MF and AF synapses at both presynaptic and postsynaptic sites. In this study, we focused on the difference in the postsynaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) between these synapses. To trace the subunit-specific trafficking of AMPARs at each synapse, GluR1 and GluR2 subunits were introduced into CA3 pyramidal neurons in hippocampal organotypic cultures using the Sindbis viral expression system. The electrophysiologically-tagged GluR2 AMPARs, produced by the viral-mediated transfer of the unedited form of GluR2 (GluR2Q), were inserted into both MF and AF postsynaptic sites in a neuronal activity-independent manner. Endogenous Ca(2+)-impermeable AMPARs at these synapses were replaced with exogenous Ca(2+)-permeable receptors, and Ca(2+) influx via the newly expressed postsynaptic AMPARs induced NMDAR-independent LTP at AF synapses. In contrast, no GluR1 AMPAR produced by the gene transfer was constitutively incorporated into AF postsynaptic sites, and only a small amount into MF postsynaptic sites. The synaptic trafficking of GluR1 AMPARs was triggered by the activity of Ca(2+)/calmodulin-dependent kinase II or high-frequency stimulation to induce LTP at AF synapses, but not at MF synapses. These results indicate that MF and AF postsynaptic sites possess distinct properties for AMPAR trafficking in CA3 pyramidal neurons.