Endosomal trafficking of AMPA-type glutamate receptors

Many different forms of synaptic plasticity have been shown to ultimately modulate the number of AMPA-type glutamate receptors at the synapse. This trafficking involves lateral movements between synaptic and extrasynaptic sites at the neuron surface, as well as vesicular transport between the plasma membrane and intracellular compartments. Several new studies have shed light on the location and regulation of AMPA-type receptor (AMPAR) endocytosis, their intracellular sorting to divergent pathways at the level of endosomes, and the mechanism and sites of receptor recycling. This review summarizes this recent data on the trafficking along the endocytic pathway, and follows the path of internalized AMPAR from endocytosis up to sites of recycling.     

Roles of distinct glutamate receptors in induction of anti-Hebbian long-term potentiation

Many glutamatergic synapses on interneurons involved in feedback inhibition in the CA1 region of the hippocampus exhibit an unusual form of long-term potentiation (LTP) that is induced only if presynaptic glutamate release occurs when the postsynaptic membrane potential is relatively hyperpolarized. We have named this phenomenon 'anti-Hebbian' LTP because it is prevented by postsynaptic depolarization during afferent activity, and hence its induction requirements are opposite to those of Hebbian NMDA receptor-dependent LTP. This symposium report addresses the roles of distinct glutamate receptors in the induction of anti-Hebbian LTP. Inwardly rectifying Ca²⁺-permeable AMPA receptors mediate fast glutamatergic signalling at synapses that exhibit this form of LTP, and they are highly likely to mediate the instructive signal that triggers the cascade leading to synapse strengthening. NMDA receptors, on the other hand, play no role, nor do they contribute substantially to synaptic transmission at synapses that exhibit anti-Hebbian LTP. Both kainate and group I metabotropic glutamate receptors are abundant in at least some interneurons in the feedback inhibitory circuit. Delineating the roles of kainate receptors has been hampered by sub-optimal pharmacological tools. As for group I metabotropic glutamate receptors, their role in anti-Hebbian LTP is permissive at the very least in some interneuron types, although an instructive role has been suggested in other forms of activity-dependent plasticity.     

Functionally different AMPA-type glutamate receptors in morphologically identified neurons in rat superficial superior colliculus.

In the superficial superior colliculus, a center of sensory processing related to visual salience, glutamate is used as a major excitatory neurotransmitter. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors include a Ca(2+)-impermeable, outwardly rectifying type (type I) and a Ca(2+)-permeable, inwardly rectifying type (type II). To study the contribution of these AMPA receptor subtypes to visual sensory processing in the superior colliculus, we investigated the expression of these two types of AMPA receptors in six morphologically identified subgroups of neurons in the superficial superior colliculus by whole-cell recording using slice preparations of young (17-23 days old) and adult (60-68 days old) rats. Both outwardly and inwardly rectifying current responses were observed to pressure applied 10 mM kainate, a non-desensitizing AMPA receptor agonist. These currents were completely abolished by the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (100 microM). The type II receptor antagonist spermine (1 mM) suppressed inwardly rectifying responses. The degree of inward rectification was correlated with the ratio of suppression by spermine, and inversely correlated with estimated Ca(2+) permeability, indicating that the degree of rectification reflects the relative amount of co-expressed type I and type II receptors. An inwardly rectifying and spermine-sensitive AMPA component of excitatory postsynaptic currents was observed, suggesting involvement of type II receptors in synaptic transmission. Morphological analysis revealed that a substantial population of horizontal cells in both young and adult rats (n=31/53 and 15/17, respectively) and all wide field multipolar cells in adult rats (n=6) showed inwardly rectifying AMPA receptor responses.From these results we suggest that type I and type II AMPA receptors are co-expressed with varying ratios in individual neurons in the rat superficial superior colliculus, and that type II receptors are abundantly expressed in most horizontal cells and wide field multipolar cells. Since these neurons are putatively GABAergic inhibitory neurons and have wide dendritic trees, type II receptors may contribute to the regulation of remote inhibitory interaction in the visual field map in the the superficial superior colliculus.     

Long-term potentiation and contextual fear conditioning increase neuronal glutamate uptake.

Induction and expression of long-term potentiation (LTP) in area CA1 of the hippocampus require the coordinated regulation of several cellular processes. We found that LTP in area CA1 was associated with an N-methyl-D-aspartate (NMDA) receptor-dependent increase in glutamate uptake. The increase in glutamate uptake was inhibited by either removal of Na+ or addition of D,L-threo-beta-hydroxyaspartate. Dihydrokainate (DHK), a specific inhibitor of the glial glutamate transporter GLT-1, did not block the increase in glutamate uptake. LTP was also associated with a translocation of the EAAC1 glutamate transporter from the cytosol to the plasma membrane. Contextual fear conditioning increased the maximum rate (Vmax) of glutamate uptake and membrane expression of EAAC1 in area CA1. These results indicate that regulation of glutamate uptake may be important for maintaining the level of synaptic strength during long-term changes in synaptic efficacy.     

Long-term potentiation in the dentate gyrus is not linked to increased extracellular glutamate concentration

Long-term potentiation (LTP) of excitatory transmission is a likely candidate for the encoding and storage of information in the mammalian brain. There is a general agreement that LTP involves an increase in synaptic strength, but the mechanisms underlying this persistent change are unclear and controversial. Synaptic efficacy may be enhanced because more transmitter glutamate is released or because postsynaptic responsiveness increases or both. The purpose of this study was to examine whether increased extracellular glutamate concentration was associated with the robust and well-characterized LTP that can be induced in the rat dentate gyrus. To favor the detection of any putative change in extracellular glutamate associated with LTP, our experimental strategy included the following features. 1) Two separate series of experiments were carried out with animals under pentobarbital or urethan anesthesia; 2) changes in extracellular concentration of glutamate were monitored continuously by microdialysis coupled to enzyme amperometry; and 3) dialysate glutamate levels and changes in the slope of excitatory postsynaptic potential evoked by activation of the perforant path were recorded precisely at the same site. Tetanic stimulation of the perforant path increased persistently test-evoked responses in the dentate gyrus (by 19 and 14% in barbiturate and urethan group, respectively), but there was no glutamate change either during or after LTP induction and no indication of increased glutamate efflux when low-frequency stimulation was applied. The results do not rule out a possible contribution of enhanced glutamate exocytosis to LTP induction and/or maintenance because such a presynaptic change may not be detectable extracellularly. However, our findings and other data supporting the notion that neurotransmitter glutamate may hardly leak out of the synaptic cleft conflict with the hypothesis that LTP could also involve a broad synaptic spillover of glutamate.     

Long-term potentiation in the hippocampus

Long-term potentiation of field and single neuronal responses recorded in various hippocampal fields is described on the basis of author's and literary data. Most of intrahippocampal and extrinsic connections in both in vivo and in vitro hippocampal preparations show this phenomenon after one or several conditioning trains of comparatively short duration (20 s or less) at various frequencies (from 10 to 400 Hz). Properties of hippocampal potentiation are described. The properties include long term persistence (hours and days) of the potentiated response, its low frequency depression, self-restoration after the depression, specificity of the potentiation for the tetanized pathway, necessity of activation of a sufficient number of neuronal elements (‘cooperativity’) to produce the potentiation, possible involvement of ‘reinforcing’ brain structures during conditioning tetanization. These properties are distinct from those of ‘usual’ short-term post-tetanic potentiation and lead to the suggestion that the neuronal mechanisms underlying long-term potentiation are similar to those underlying memory and behavioralconditioned reflex. Neurophysiological mechanisms of long-term potentiation are discussed. The main mechanism consists in an increase in efficacy of excitatory synapses as shown by various methods including intracellular recording and quantal analysis. The latter favours presynaptic localization of the changes of synaptic efficacy showing increase in the number of transmitter quanta released per presynaptic impulse. However, changes in the number of subsynaptic receptors or localized changes in dendritic postsynaptic membrane are not excluded. Biochemical studies indicate the increase in transmitter release and calcium-dependent phosphorylation of pyruvate dehydrogenase after tetanization. Instances of persistent response facilitations at other levels of the vertebrate central nervous system (especially at neocortical level) are considered and compared with hippocampal long-term potentiation.

It is suggested that modifiable excitatory synapses necessary for learning have been identified in studies of long-term potentiation. These synapses are presumably modified as a result of close sequential activation of the following three structures: excitatory presynaptic fibers, the postsynaptic neuron and a ‘reinforcing’ brain system.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The Edinger-Westphal nucleus (EW) in birds is responsible for the control of pupil constriction, accommodation, and choroidal blood flow. The activation of EW neurons is mediated by the neurotransmitter glutamate, in large part through AMPA-type glutamate receptors (GluRs), whose behavior varies according to the subunit composition. We investigated the developmental expression of the GluR subunits in EW of the chick (Gallus gallus) using immunohistochemistry on tissue from embryonic days 10 through 20 (E10–E20). Of the three antibodies used, one recognized the GluR1 subunit, another the GluR4 subunit, and the third recognized a sequence common to GluR2 and GluR3 subunits. No immunolabeling of EW neurons for any GluR subunits was observed prior to E12, although immunolabeling was seen in somatic oculomotor prior to E12. At E12, immunoreactivity for each of the three antibodies was in only approximately 2% of EW neurons. By E14, the abundance of GluR1+ perikarya in EW had increased to 13%, and for GluR2/3 had increased to 48%. The perikaryal abundance of the immunoreactivity for GluR1 and GluR2/3 declined to 3% and 23%, respectively, by E16. At E14, 33% of EW neurons immunolabeled for GluR4, and their frequency increased to 43% by E16, and remained at that approximate percentage through hatching. The increased expression of GluR1 and GluR4 in EW at E14 coincides with the reported onset of the expression of the calcium-binding protein parvalbumin, and the calcium currents associated with AMPA receptors formed by these two subunits may play a role in the occurrence of parvalbumin expression.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.     

Presynaptic group 1 metabotropic glutamate receptors may contribute to the expression of long-term potentiation in the hippocampal CA1 region

In this study, we investigated the possible contribution of presynaptic group 1 metabotropic glutamate receptor activation to changes in synaptic efficacy by means of analysis of glutamate release in hippocampal synaptosomes. Data were interpreted in the context of group 1 metabotropic glutamate receptor involvement in synaptic plasticity in the CA1 region of freely moving rats. In synaptosomes, 3,5-dihydroxyphenylglycine enhanced diacylglycerol formation and facilitated vesicular Ca(2+)-dependent glutamate release, whereas trans-azetidine-2,4-dicarboxylic acid had no effect on these processes. Trans-azetidine-2,4-dicarboxylic acid enhanced glutamate release, but in a Ca(2+)-independent manner. This effect was mimicked by the L-glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid. (R,S)-alpha-Methyl-4-carboxyphenylglycine blocked the effects of 3,5-dihydroxyphenylglycine, but not trans-azetidine-2,4-dicarboxylic acid in synaptosomes. Short-term potentiation (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) was induced in the CA1 region in vivo. The metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-2,3-dicarboxylic acid, or the group 1 metabotropic glutamate receptor agonists, 3,5-dihydroxyphenylglycine and trans-azetidine-2,4-dicarboxylic acid, dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted > 24 h. The facilitation was inhibited by the metabotropic glutamate receptor antagonist, (R,S)-alpha-methyl-4-carboxyphenylglycine, and the group 1 metabotropic glutamate receptor antagonist, (S)-4-carboxy-phenylglycine, but not by the group 2 metabotropic glutamate receptor antagonist, (R,S)-alpha-methylserine-O-phosphate monophenyl ester. L-Trans-pyrrolidine-2,4-dicarboxylic acid dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted < 4 h. These data suggest that activation of group 1 metabotropic glutamate receptors results in presynaptic modulation of glutamate release. This effect may contribute to group 1 metabotropic glutamate modulation of the expression of long-term potentiation in vivo.     

Activation of group II metabotropic glutamate receptors results in long-term potentiation following preconditioning stimulation in the dentate gyrus.

The role of group II metabotropic glutamate receptors in the induction/expression of long-term potentiation has been investigated in the medial perforant path of the outer (infrapyramidal) blade of the rat dentate gyrus in vitro. Activation of group II metabotropic glutamate receptors by perfusion of the selective agonist LY354740 did not induce long-term potentiation or long-term depression in control. However, LY354740, applied following the induction of long-term potentiation by high frequency stimulation, resulted in additional long-term potentiation. LY354740 was only found to cause additional long-term potentiation if the pre-existing high frequency stimulation-induced long-term potentiation was sub-maximal. Although activation of metabotropic glutamate receptors was not required for induction of high frequency stimulation-induced long-term potentiation, activation of both group I and group II metabotropic glutamate receptors was required during high frequency stimulation-induced long-term potentiation in order for subsequent application of LY354740 to result in additional long-term potentiation. Thus, the long-term potentiation caused by application of LY354740 following high frequency-induced long-term potentiation was prevented if the high frequency stimulation was given in the presence of (S)-alpha-methyl-4-carboxyphenylglycine or the selective group I or group II metabotropic glutamate receptor antagonists 1-aminoindan-1,5-dicarboxylic acid or (2S)-alpha-ethylglutamic acid respectively. The long-term potentiation caused by LY354740 was also dependent upon activation of N-methyl-D-aspartate receptors during the high frequency stimulation, being blocked if high frequency stimulation was given in the presence of the N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid. The long-term potentiation resulting from activation of group II metabotropic glutamate receptors could be due either to the enhancement of the expression level of the high frequency stimulation-induced long-term potentiation, or alternatively, to a direct novel induction of long-term potentiation. In either theory, the long-term potentiation resulting from activation of group II metabotropic glutamate receptors is dependent upon prestimulation of group I and group II metabotropic glutamate receptors and N-methyl-D-aspartate receptors during the 'preconditioning high frequency stimulation'.     

Transient translocation of protein kinase Cgamma in hippocampal long-term potentiation depends on activation of metabotropic glutamate receptors.

Protein kinase C has been implicated in long-term regulation of cellular functions including induction and maintenance of hippocampal long-term potentiation. In the present study the time-course of long-term potentiation-induced translocation of Ca(2+)-dependent protein kinase C isoenzymes (PKCalpha/beta and PKCgamma) was investigated. Quantitative immunoblot analysis was used to measure translocation of these isoenzymes between cytosolic, membrane-associated and membrane-inserted fraction at 5, 15 and 60 min after induction of long-term potentiation in the dentate gyrus in vivo. To investigate the involvement of metabotropic glutamate receptors in protein kinase C regulation during long-term potentiation induction, additional animals were treated before tetanization with (R,S)-alpha-methyl-4-carboxyphenylglycine, an antagonist of metabotropic glutamate receptors. Brief tetanic stimulation of the perforant path resulted in a 100-150% increase in the population spike amplitude in response to test stimuli 5, 15 or 60 min after stimulation in both untreated and (R,S)-alpha-methyl-4-carboxyphenylglycine-treated animals. Only those rats showing clear potentiation were selected for further biochemical analysis of the potentiated dentate gyrus. Five minutes after high-frequency stimulation the subcellular distribution of all studied protein kinase C isoenzymes was unchanged compared with controls. PKC-gamma translocated into the cytosol 15 min after tetanization and this redistribution was blocked by (R,S)-alpha-methyl-4-carboxyphenylgly-cine pretreatment. By contrast, PKC alpha/beta levels increased in the cytosolic fraction only 60 min after tetanization, but in a (R,S)-alpha-methyl-4-carboxyphenylglycine-independent manner. In an additional set of experiments it was shown that (R,S)-alpha-methyl-4-carboxyphenylglycine alone applied intraventricularly had no effect on the subcellular distribution of the studied isoenzymes. The data suggest that PKCalpha/beta and PKCgamma are activated during different post-tetanic phases and metabotropic glutamate receptor activation might be essential for tetanus-induced translocation of postsynaptic PKCgamma only.     

Long-term potentiation in the dentate gyrus: induction and increased glutamate release are blocked by D(-)aminophosphonovalerate

D(-)Aminophosphonovalerate, a specific antagonist of the N-methyl-D-aspartate subtype of glutamate receptor, was perfused through a push-pull cannula into the dentate gyrus of rats anaesthetized with urethan in order to observe its effect on the induction and maintenance of long-term potentiation and on the increase in release of endogenous glutamate associated with long-term potentiation. The amplitude of the population spike evoked by single test shocks to the perforant path was significantly depressed by 100 microM D(-)aminophosphonovalerate, but there was a minimal effect on the slope of the population excitatory postsynaptic potential, or on the concentration of glutamate released into the perfusate. A brief high-intensity tetanus given to the perforant path while D(-)aminophosphono-valerate was being perfused failed to induce long-term potentiation or the sustained increase in glutamate release associated with long-term potentiation. Short-term post-tetanic potentiation was not affected. After wash-out of D(-)aminophosphonovalerate, a second high-frequency train produced both long-term potentiation and an increase in glutamate release which was sustained for the subsequent 1 h period of observation. D(-)Aminophosphonovalerate did not suppress long-term potentiation once it had been induced. D(-)Aminophosphonovalerate (100 microM) did not itself affect in vivo release of glutamate. However, in a separate series of in vitro experiments, D(-)aminophosphonovalerate at concentrations of 50 microM and above was found to depress the Ca2+ -dependent, K+-stimulated release of preloaded [14C]-glutamate from dentate slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term potentiation and depression in the cerebral neocortex

Long-term potentiation of synaptic efficacy following tetanic synaptic inputs was described originally in the hippocampus, and it has been studied extensively based on the hypothesis that it represents a synaptic model of learning and memory in the brain. In the cerebral neocortex, studies on LTP have burgeoned later, and have progressed less rapidly than those in the hippocampus. Recently, however, experimental data describing the phenomenology and the mechanisms underlying LTP have accumulated in the neocortex, particularly in the visual, somatosensory, and motor cortices. In the developing visual cortex, LTP has been induced by afferent tetanic stimulation at relatively low frequencies, for long duration. Thus, particular attention has been given to parameters of the tetanus optimal for the induction of cortical LTP, and the differences between these and those effective in inducing hippocampal LTP have been reviewed. In the motor cortex, the associative LTP following combined activation of separate sites as well as homosynaptic LTP following activation of single pathways have been reported and these types of synaptic plasticity have been suggested as being a basis for a certain type of motor learning. Long-lasting depression (LTD) of synaptic efficacy also has been reported in the developing visual cortex and suggested as a neural basis for experience-dependent modifications of visual cortical neurons. LTD has been found in other areas of the neocortex as well, although the probability of its induction is relatively low and its functional significance is not yet clear. Among the possible mechanisms for the induction of LTP and LTD, those including the involvement of NMDA receptors, protein kinase C, Ca2+/calmodulin-dependent kinase II, and membrane-associated cytoskeletal proteins have been reviewed, although the results obtained so far are only fragmentary and are premature for definitive conclusions to be drawn.     

Long-term potentiation in the optic tectum of rainbow trout

We examined synaptic plasticity in the optic tectum of rainbow trout by extracellular recordings. We found that the field-excitatory postsynaptic potential in the retinotectal synapses was potentiated by repetitive stimuli of 1.0 Hz for 20 s to the retinotectal afferents. The long-term potentiation (LTP) developed slowly, and was maintained for at least 2 h. Applications of an antagonist for N-methyl-D-aspartic acid (NMDA) receptors or Mg²⁺-free saline showed that activation of NMDA receptors was required to form the LTP beyond the induction period. The present findings indicate that presynaptic stimulation in the retinotectal synapses causes LTP mediated by NMDA receptors in the optic tectum of rainbow trout.     

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.