Glutamate receptor IIA and Glutamate receptor IIB

EVOLUTIONARY HOMOLOGS

Table of contents

Signaling downstream of glutamate receptors

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and AMPA receptors. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na+ through these ion channels. Binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brings about changes in a guanine-nucleotide-binding protein (Galpha[il]) that inhibit pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect is observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and is also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibits forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins are activated. Moreover, both Gbetagamma blockage and co-precipitation experiments demonstrate that the modulation of the Gi protein arises from the association of Galpha(il) with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity (Wang, 1997).

The AMPA receptor, ubiquitous in brain, is termed 'ionotropic' because it gates an ion channel directly. An AMPA receptor can also modulate a G-protein to gate an ion channel indirectly. Glutamate applied to a retinal ganglion cell briefly suppresses the inward current through a cGMP-gated channel. AMPA and kainate also suppress the current, an effect that is blocked both by their general antagonist CNQX and also by the relatively specific AMPA receptor antagonist GYKI-52466. Neither NMDA nor agonists of metabotropic glutamate receptors are effective. The AMPA-induced suppression of the cGMP-gated current is blocked when the patch pipette includes GDP-beta-S, whereas the suppression is irreversible when the pipette contains GTP-gamma-S. This suggests a G-protein mediator, and, consistent with this, pertussis toxin blocks the current suppression. Nitric oxide (NO) donors induce the current suppressed by AMPA, and phosphodiesterase inhibitors prevent the suppression. Apparently, the AMPA receptor can exhibit a 'metabotropic' activity that allows it to antagonize excitation evoked by NO (Kawai, 1999).

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. The ionotropic glutamate receptors are classified into two groups: NMDA (N-methyl-D-aspartate) receptors and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors. The AMPA receptor is a ligand-gated cation channel that mediates the fast component of excitatory postsynaptic currents in the central nervous system. AMPA receptors function not only as ion channels but also as cell-surface signal transducers by means of their interaction with the Src-family non-receptor protein tyrosine kinase Lyn. In the cerebellum, Lyn is physically associated with the AMPA receptor and is rapidly activated following stimulation of the receptor. Activation of Lyn is independent of Ca2+ and Na+ influx through AMPA receptors. As a result of Lyn activation, the mitogen-activated protein kinase (MAPK) signaling pathway is activated, and the expression of brain-derived neurotrophic factor (BDNF) messenger RNA is increased in a Lyn-kinase-dependent manner. Thus, AMPA receptors generate intracellular signals from the cell surface to the nucleus through the Lyn-MAPK pathway, which may contribute to synaptic plasticity by regulating the expression of BDNF (Hayashi, 1999).

Ca2+-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody, the ability of AMPA receptors to activate the MAPK cascade was addressed. In the presence of cyclothiazide, AMPA causes a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca2+ channels) Ca2+-dependent activation of MAPK through MAPK kinase (MEK). This activation is blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation reveals an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca2+/calmodulin-dependent kinase II. Application of AMPA to rat cerebral cortical neurons has been shown to lead to a rapid increase in Ras activity and activation of MAPK. Ras-dependent activation of MAPK is usually associated with seven transmembrane receptors that couple to heterotrimeric G-proteins. AMPA activates ERK2 (p42) by causing a Ca2+-dependent association of G-protein betagamma subunits, probably Gi, with a Ras, Raf kinase, MEK complex. This novel involvement of a heterotrimeric G-protein in ionotropic AMPA receptor signaling was examined. Striatal neurons were pretreated with pertussis toxin (PTX) or PBS vehicle for 24 hr before experiments with AMPA/cyclothiazide. PTX treatment abolishes AMPA receptor activation of MAPK, indicating a role for a Gi or Go-type G-protein in the activation of MAPK by AMPA receptors in striatal neurons. Similarly, kainate activates MAPK in a PI 3-kinase-dependent manner. AMPA receptor stimulation leads to a Ca2+-dependent phosphorylation of the nuclear transcription factor CREB, which can be prevented by inhibitors of MEK or PI 3-kinase. These results indicate that Ca2+-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum (Perkinton, 1999).

Thus, although the specific protein-protein interactions that lead to activation of the Ras-MAPK pathway by AMPA receptors are not currently known, it seems reasonable to propose that AMPA receptor-evoked rises in cytosolic Ca2+ may trigger activation of PI 3-kinase: then, recruitment of the lipid kinase to the MAPK cascade may, as is the case with seven-transmembrane Gi/Go-type G-protein linked receptors, be orchestrated by free G betagamma subunits. The specific exchange factors regulating Ras activity after AMPA receptor stimulation also remain to be determined. An involvement of the neuron-specific guanine nucleotide exchange factor, Ras-GRF, seems plausible because it has recently been demonstrated that Ras-GRF can be activated in response to increases in intracellular Ca2+ and/or free G-protein betagamma subunits that induce phosphorylation of Ras-GRF by as yet unknown kinases. However, Ca2+/calmodulin-dependent activation of Ras-GRF does not appear to involve PTKs, thus, the results indicating that tyrosine phosphorylation may be an important step in AMPA receptor activation of MAP kinase suggests that additional Ca2+-dependent routes to Ras may be activated. It has been shown that CaM-KII can phosphorylate AMPA receptor subunits (Mammen et al., 1997), resulting in enhanced receptor currents, and this has been implicated in the strengthening of postsynaptic responses associated with synaptic plasticity. Selective inhibition of CaM-KII activity substantially reduces AMPA/cyclothiazide-evoked activation of MAPK without altering Ca2+ influx through the receptor. These data indicate that CaM-KII can be a positive modulator of AMPA receptor signaling but that in the presence of cyclothiazide the kinase probably regulates AMPA receptor-mediated MAPK activation at a point downstream of Ca2+ entry (Perkinton, 1999 and references).

alpha and ßCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/ß ratio shifts toward alpha during increased activity and ß during decreased activity. The swing in ratio is ~5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca²⁺ signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving ßCaMKII and possibly further downstream effects of ßCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or ßCaMKII results in opposing effects on unitary synaptic strength as well as mEPSC frequency that can account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity (Thiagarajan, 2002).

Calcium/calmodulin-dependent protein kinase II (CaMKII) is expressed at high levels in the central nervous system, particularly in the hippocampus, where it constitutes ~2% of total protein. As a holoenzyme, neuronal CaMKII is made up of 6-12 subunits, primarily the 52 kDa alpha isoform and the 60 kDa ß isoform. The subunits of the holoenzyme are held together by association domains in their C-terminals, which form a central globular structure from which the N-terminals extend radially. The N-terminal contains the catalytic sites of the kinase as well as the autoinhibitory domains that bind to the catalytic sites in the basal state. The binding of Ca²⁺/calmodulin releases this autoinhibition, allowing phosphorylation to take place at a critical threonine residue, Thr²⁸⁶ in alpha and Thr²⁸⁷ in ß. This autophosphorylation allows the molecular memory of a transient Ca²⁺ signal to greatly outlast the duration of the Ca²⁺ transient itself, a property that endows CaMKII with the ability to decode Ca²⁺ signals in a frequency-dependent manner (Thiagarajan, 2002 and references therein).

Immunoprecipitation with subunit-specific antibodies indicates that the majority of the CaMKII holoenzymes are alpha/ß heteromers with variable subunit ratios, although some alpha homomers can also be found. Why have two isoforms? One significant distinction between the alpha and ß isoforms lies in their sharply different affinity for calmodulin. Half-maximal autophosphorylation is achieved at 130 nM calmodulin for alphaCaMKII and at 15 nM calmodulin for ßCaMKII. Due to this difference, the two isoforms have different sensitivities to Ca²⁺ signals under nonsaturating levels of calmodulin. alphaCaMKII is selective for higher levels of Ca²⁺ signals, while ßCaMKII has better sensitivity to lower levels of signal. When the two isoforms are combined in a heteromer, the response to Ca²⁺ signals has been found to depend on the ratio of alpha to ß subunits. Consequently, activity-dependent regulation of alpha- and ßCaMKII expression could provide a mechanism of tuning neuronal responses to different levels of activity. This is an intriguing possibility that raises several fundamental questions. Does the cell regulate the ratio of alpha to ß in an activity-dependent manner? And if so, what pathways of synaptic activity might control the regulation of alpha- and ßCaMKII? Could their regulation be coupled? What would be the consequence of such a regulation for synaptic transmission? This study uses a combination of immunotechniques and electrophysiology to address these issues. The data show that alpha- and ßCaMKII are inversely regulated by activity in a manner that may help tune CaMKII to changing levels of Ca²⁺ signal. Furthermore, tilting the ratio toward alpha or ß results in opposing effects on unitary synaptic strength and mEPSC frequency and has functional significance for both activity-dependent plasticity and homeostasis (Thiagarajan, 2002).

In excitatory neurons, treatment with TTX to block action potentials decreases the levels of alphaCaMKII and raises ßCaMKII, both in cell bodies and at synapses. Conversely, exposure to bicucculine to prevent inhibitory transmission and increase firing increases alphaCaMKII and decreased ßCaMKII. Thus, the changes in alphaCaMKII correlated positively with changes in electrical activity, while changes in ßCaMKII correlated negatively. This inverse regulation gives rise to ~5-fold changes in the alpha:ß ratio between the extremes of TTX or BIC treatment, while the sum total of these isoforms remained relatively unchanged, varying only 1.0- to 1.3-fold (for assumed values of the basal alpha/ß ratio ranging between 1:1 and 3:1). One may speculate that the inverse changes in isoform levels support the widest variation in isoform ratio consistent with holding fixed the total amount of enzyme (possibly important for structural reasons). That inhibitory neurons, which lack immunoreactivity for alphaCaMKII, fail to show regulation of ß with altered activity also fits with a pattern in which the overall level of CaMKII is tightly regulated (Thiagarajan, 2002).

Changes in the balance between alpha and ß isoforms predicts interesting functional consequences. ßCaMKII has been shown to exhibit a much higher affinity than alphaCaMKII for Ca²⁺/CaM. If the changes in the overall alpha:ß ratio between the opposite conditions of TTX and BIC are indicative of the subunit composition of the CaMKII holoenzyme, this would result in a much higher affinity of the holoenzyme for Ca²⁺/CaM. Assuming a ~9-fold difference in CaM affinity of ßCaMKII relative to alphaCaMKII, a rough calculation predicts a ~2-fold variation in the holoenzyme affinity for Ca²⁺/CaM. The increased sensitivity of the holoenzyme to CaM with decreased activity would serve as a homeostatic mechanism to confer responsiveness to weaker Ca²⁺ signals (Thiagarajan, 2002).

It first came as a surprise that activity-dependent changes in alpha and ß isoforms arose from largely distinct pathways, involving different glutamate receptors: levels of alphaCaMKII (but not ß) are strongly influenced by NMDAR activity; in contrast, ßCaMKII was strongly affected by AMPAR activity. These new findings make sense if put in context of previous studies on neuronal CaMKII. The strong reduction of alphaCaMKII but sparing of ß by blockade of NMDA receptors can be interpreted in light of several related observations: (1) multimeric CaMKII takes the form of alpha homomers as well as alpha/ß heteromultimers; (2) NMDAR-dependent changes in the abundance of alphaCaMKII can be detected as soon as 5 min after stimulation in hippocampal slices, consistent with a localized dendritic translation of alphaCamKII mRNA; (3) ßCaMKII mRNA is absent in dendrites, leaving alpha homomers as the only enzyme species that could be formed there. Taken together with these observations, these findings are consistent with a simple model wherein Ca²⁺ entry through NMDARs in the dendrites regulates alphaCaMKII homomers locally, on a fast time scale, with little or no control of ß (Thiagarajan, 2002).

The observation that levels of ßCaMKII strongly increased in response to blockade of AMPA receptors, not NMDARs, suggested that regulation of ß may be quite different than proposed for alpha alone. Under various pharmacological conditions, the pattern of changes observed in ßCaMKII was always consistent with an inverse relationship with AMPAR activity. Because ß transcripts are restricted to the cell body, and changes in ßCaMKII occur only slowly (evident only on time scales >1 hr), the regulation by AMPARs is likely to occur at the level of nuclear transcription. Thus, AMPAR-mediated depolarization could work through recruitment of voltage-gated Ca²⁺ channels and regulation of nuclear transcription factors. Regulation in or near the nucleus makes additional sense for the linkage between increased ßCaMKII and the downregulation of alpha that was observed in transfection studies. All considerations seem consistent with the following working hypothesis: ßCaMKII levels are regulated in the cell body, downstream of AMPA receptor activity, leading to a reciprocal regulation of alpha and thus the formation of alpha/ß heteromers of variable subunit ratio (Thiagarajan, 2002).

This scheme invokes the inter-relationship between levels of ß and alpha that was directly observed in the transfection experiments. Another possibility, not mutually exclusive, is that AMPAR block decreases postsynaptic depolarization and thereby reduces Ca²⁺ entry through NMDAR, leading to a fall in the alpha isoform (Thiagarajan, 2002).

Conceptual distinctions have been drawn between synaptic homeostasis, negative feedback regulation thought of as neuron wide, and forms of synaptic plasticity such as LTP, which can be self-reinforcing and synapse specific. Both kinds of regulation may be strongly impacted by inverse changes in the abundance of alpha- and ßCaMKII. For example, increased activity, and consequent elevation of the alpha/ß ratio would decrease the Ca²⁺/CaM sensitivity of CaMKII in a homeostatic manner. This can be viewed as 'input tuning', wherein the holoenzyme is adjusted appropriately to the ambient level of activity. The threshold for the induction of LTP, which already is high, would be further raised, thereby changing the rules governing synaptic plasticity . However, increasing the alpha/ß ratio may also change the cellular localization of CaMKII, promoting alphaCaMKII expression at specific subsynaptic sites where it could contribute to LTP. Understanding the full implications for plasticity and metaplasticity will become easier once more is known about how the subunit composition of CaMKII affects its cellular localization and degree of autophosphorylation and how alterations in the alpha/ß ratio and its downstream effects unfold over time scales ranging from minutes to days (Thiagarajan, 2002).

Altering the levels of alpha and ß causes striking changes in both mini size and frequency. Once again, the overall change in synaptic function cannot be neatly pigeonholed into strict categories of 'synaptic homeostasis' or 'synaptic plasticity' alone. Increases in the alpha/ß ratio accentuates the contribution of individual synaptic events (augmented mini area), while also tending to decrease the number of quanta received per unit time (lowered mini frequency), thus keeping the total synaptic drive within reasonable bounds. Evidently, simple biochemical changes can induce a powerful combination of self-reinforcing local changes, but negative feedback regulation over the neuron as a whole (Thiagarajan, 2002).

NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. The Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with either RNAi or dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines (Tolias, 2005).

Glutamate receptors and adult neural stem cells

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. This study demonstrates that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca_v1.2/1.3 (L-type) Ca²⁺ channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an 'excitation-neurogenesis coupling rule' within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories (Deisseroth, 2004).

Using an array of approaches, the coupling of excitation to neurogenesis in proliferating adult-derived NPCs was studied both in vitro and in vivo. Adult neurogenesis is potently enhanced by excitatory stimuli and involves Ca_v1.2/1.3 channels and NMDA receptors. These Ca²⁺ influx pathways are located on the proliferating NPCs, allowing them to directly sense and process excitatory stimuli. No effect of excitation was found on the extent of differentiation in individual cells (measured by extent of MAP2ab expression in the NPC-derived neurons) nor were effects observed on proliferative rate or fraction, survival, or apoptosis. Instead, excitation increased the fraction of NPC progeny that were neurons, both in vitro and in vivo, and total neuron number was increased as well. The Ca²⁺ signal in NPCs leads to rapid induction of a proneural gene expression pattern involving the bHLH genes HES1, Id2, and NeuroD, and the resulting cells become fully functional neurons defined by neuronal morphology, expression of neuronal structural proteins (MAP2ab and Doublecortin), expression of neuronal TTX-sensitive voltage-gated Na⁺ channels, and synaptic incorporation into active neural circuits. A monotonically increasing function characterizes excitation-neurogenesis coupling, and incorporation of this relationship into a layered Hebbian neural network suggests surprising advantages for both the clearance of old memories and the storage of new memories. Taken together, these results provide a new experimental and theoretical framework for further investigation of adult excitation-neurogenesis coupling (Deisseroth, 2004).

In the hippocampal formation, neural stem cells exist either within the adjacent ventricular zone or within the subgranular zone proper at the margin between the granule cell layer and the hilus, where proliferative activity is most robust. These cells do not express neuronal markers but proliferate and produce dividing progeny that incrementally commit to differentiated fates (such as the neuronal lineage) over successive cell divisions. Native NPC populations in vivo are therefore heterogenous with regard to lineage potential, and markers are not available that distinguish between the multipotent stem cell and the subtly committed yet proliferative progenitor cell. Excitation may therefore act on either or both types of proliferating precursor, in vitro and in vivo. The functional consequences of coupling excitation to insertion of new neurons for the neural network, however, is independent of which precursor cell types respond to excitation (Deisseroth, 2004).

The enhancement of hippocampal neurogenesis by behavioral stimuli such as environmental enrichment and running may, at least in part, be implemented at the molecular level by excitation-neurogenesis coupling. Notably, running and environmental enrichment increase adult neurogenesis in the hippocampus but not in the subventricular zone. Of course, not every neurogenic region in the brain need follow the excitation-neurogenesis coupling rule outlined here. An activity rule appropriate for the unique information processing or storage function of that brain region might be expected to operate. In this context, it is interesting to note that, while subventricular zone/olfactory bulb precursor neurogenesis is not enhanced by behavioral activity, proliferation and survival in this system can be influenced by olfactory sensory stimuli. This suggests that a different form of activity rule, appropriate for that local circuit, may govern olfactory bulb neurogenesis (Deisseroth, 2004).

AMPA receptors: activity modification of neural structure

The influence of synaptically released glutamate on postsynaptic structure was investigated by comparing the effects of deafferentation, receptor antagonists and blockers of glutamate release in hippocampal slice cultures. Spine density and length of CA1 pyramidal cells decrease after transection of Schaffer collaterals and after application of AMPA receptor antagonists or botulinum toxin to unlesioned cultures. Loss of spines induced by lesion or by botulinum toxin is prevented by simultaneous AMPA application. Tetrodotoxin does not affect spine density. Synaptically released glutamate thus exerts a trophic effect on spines by acting at AMPA receptors. It is concluded that AMPA receptor activation by spontaneous vesicular glutamate release is sufficient to maintain dendritic spines (McKenney, 1999).

The classically conditioned vertebrate eye-blink response is a model in which to study neuronal mechanisms of learning and memory. In this paradigm, an eye-blink reflex in response to a tone can be evoked when the tone is repeatedly paired with an air puff to the cornea that normally elicits the blink response. A neural correlate of this response recorded in the abducens nerve can be conditioned entirely in vitro using an isolated brainstem-cerebellum preparation from the turtle by pairing trigeminal and auditory nerve stimulation. Conditioning requires that the paired stimuli occur within a narrow temporal window of <100 msec. Conditioning is blocked by a NMDA receptor antagonist. Moreover, there is a significant positive correlation between the levels of conditioning and greater immunoreactivity with the glutamate receptor 4 (GluR4) AMPA receptor subunit in the abducens motor nuclei, but not with NMDAR1 or GluR1. It is concluded that in vitro classical conditioning of an abducens nerve eye-blink response is generated by NMDA receptor-mediated mechanisms that may act to modify the AMPA receptor by increasing GluR4 subunits in auditory nerve synapses (Keifer, 2001).

Both theoretical and experimental work have suggested that central neurons compensate for changes in excitatory synaptic input in order to maintain a relatively constant output. Inhibition of excitatory synaptic transmission in cultured spinal neurons leads to an increase in mEPSC amplitudes, accompanied by an equivalent increase in the accumulation of AMPA receptors at synapses. Conversely, increasing excitatory synaptic activity leads to a decrease in synaptic AMPA receptors and a decline in mEPSC amplitude. The time course of this synaptic remodeling is slow, similar to the metabolic half-life of neuronal AMPA receptors. Moreover, inhibiting excitatory synaptic transmission significantly prolongs the half-life of the AMPA receptor subunit GluR1, suggesting that synaptic activity modulates the size of the mEPSC by regulating the turnover of postsynaptic AMPA receptors (O'Brien, 1998).

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. This study identifies palmitate cycling on PSD-95 at the synapse; palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. Rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity (El-Husseini, 2002).

AMPA receptors: long-term potentiation and depression

The ability of central glutamatergic synapses to change their strength in response to the intensity of synaptic input, which occurs, for example, in long-term potentiation (LTP), is thought to provide a cellular basis for memory formation and learning. LTP in the CA1 field of the hippocampus requires activation of Ca2+/calmodulin-kinase II (CaM-KII), which phosphorylates Ser-831 in the GluR1 subunit of the AMPA glutamate receptor (AMPA-R), and this activation/phosphorylation is thought to be a postsynaptic mechanism in LTP. In this study, a molecular mechanism has been identified by which CaM-KII potentiates AMPA-Rs. Coexpression in HEK-293 cells of activated CaM-KII with GluR1 does not affect the glutamate affinity of the receptor, the kinetics of desensitization and recovery, channel rectification, open probability, or gating. Single-channel recordings identify multiple conductance states for GluR1, and coexpression with CaM-KII or a mutation of Ser-831 to Asp increases the contribution of the higher conductance states. These results indicate that CaM-KII can mediate plasticity at glutamatergic synapses by increasing single-channel conductance of existing functional AMPA-Rs or by recruiting new high-conductance-state AMPA-Rs (Derkach, 1999).

The mechanisms responsible for enhanced transmission during long-term potentiation (LTP) at CA1 hippocampal synapses remain elusive. Several popular models for LTP expression propose an increase in 'use' of existing synaptic elements, such as increased probability of transmitter release or increased opening of postsynaptic receptors. To test these models directly, a GluR2 knockout mouse was studied in which AMPA receptor transmission was rendered sensitive to a use-dependent block by polyamine compounds. This method can detect increases during manipulations affecting transmitter release or AMPA receptor channel open time and probability, however, no such changes are seen to occur during LTP. These results indicate that the recruitment of new AMPA receptors and/or an increase in the conductance of these receptors is responsible for the expression of CA1 LTP (Mainen, 1998).

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, mice were generated that lack this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices is markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation are normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibit increased mortality, and those surviving show reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior (Jia, 1996).

Gene-targeted mice lacking the AMPA receptor subunit GluR-A exhibit normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice show a reduction in function of AMPA receptors, with the remaining receptors preferentially targeted to synapses. Immunocytochemistry in the hippocampus of GluR-A-/- mice relative to wild type reveals a redistribution of the GluR-B subunit in pyramidal and dentate granule cells with increased staining over the somata (stratum pyramidale) and decreased staining in the basal (stratum oriens) and apical (stratum radiatum) dendrites. The altered GluR-B localization upon lack of GluR-A may indicate that the edited GluR-B subunit requires a partner for assembly or dendritic targeting of GluR-B-containing AMPA receptors. A substantial amount of GluR-B remains in the stratum lacunosum-moleculare (possibly in the form of GluR-B/C heteromeric channels) in synapses at the distalmost part of the apical dendrites of CA1 and CA3 pyramidal neurons. In GluR-A-/- mice, the CA1 soma-patch currents are strongly reduced, but glutamatergic synaptic currents are unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials are as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) is absent in CA3 to CA1 synapses, but spatial learning in the water maze is not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory (Zamanillo, 1999).

This finding adds to a growing number of examples of a dichotomy between LTP and learning. One explanation could be that mice may use extrahippocampal structures to solve the Morris water maze. It is possible that LTP, although not critical for the type of reference memory test used to solve the Morris water maze, could be important in spatial tasks that involve only episodic or working memory. Alternatively, learning may be associated with LTP at a degree of synaptic involvement that is too small to be detected with conventional electrophysiological field recordings. Furthermore, the spatial task might not require LTP in Schaffer collateral-CA1 synapses. Notably, a genetically engineered CA1 NMDA receptor deficiency also generates LTP deficiency, but learning in the water maze is impaired. One explanation for the difference in learning might be that the induction phase of LTP is impaired in the NMDA receptor but not in the AMPA receptor mutant. During this phase the spinous Ca2+ transients affect numerous signaling pathways, which might be essential for memory acquisition. In this context, it might be asked how the fEPSP-LTP phenomenon is related to normal physiology in the hippocampus. The highly synchronous ensemble activity of CA3 pyramidal neurons required to induce standard fEPSP-LTP may not normally occur. In summary, adult hippocampal LTP depends on the number and subunit composition of AMPA receptors. Therefore, in adult animals, LTP appears to be essentially a postsynaptic mechanism. However, this particular form of synapse modifiability in CA1 is not required for a reference memory test (Zamanillo, 1999 and references).

To monitor changes in AMPA receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) is functional and is transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP is intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induces a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events require synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediated transmission observed during long-term potentiation and activity-dependent synaptic maturation (Shi, 1999).

These results build on a number of studies suggesting that the delivery of AMPA receptors to synapses contributes to activity-dependent plasticity. Inhibition of membrane fusion processes in the postsynaptic cell blocks the action of LTP. Furthermore, the COOH-termini of AMPA receptor subunits GluR2 and GluR4c bind N-ethylmaleimide-sensitive fusion protein, a protein involved in membrane fusion processes. Vesicular organelles, possibly undergoing exocytosis and endocytosis, have been detected with electron microcopy in spines. And last, dendrites can display a calcium-evoked exocytosis of trans-Golgi-derived organelles that is mediated by the calcium/calmodulin-dependent protein kinase II, an enzyme thought to mediate LTP. Other postsynaptic mechanisms, such as an increase in conductance of AMPA receptors, may also occur in parallel. These results also do not rule out a contribution by presynaptic modifications. In addition to the spine delivery of GluR1-GFP, tetanic stimulation induces the formation of clusters of the tagged receptor within dendrites. These structures may be related to the spine apparatus, membranous structures at the base of spines that appear to contain AMPA receptors. The entry of calcium through synaptic NMDA receptors may cause nucleation of AMPA receptor-containing membranes close to active synapses. Once formed, such sites may serve several functions. These sites may replenish those receptors delivered to spines during plasticity. Additionally, they may serve as a 'synaptic tag', providing a docking site for AMPA receptors synthesized at distant sites. Last, they could provide a site for local AMPA receptor synthesis. In these capacities, such clusters could represent a structural modification serving as a long-lasting memory mechanism (Shi, 1999 and references).

The second messenger pathways linking receptor activation at the membrane to changes in the nucleus are just beginning to be unraveled in neurons. The work presented here attempts to identify in striatal neurons the pathways that mediate cAMP response element-binding protein (CREB) phosphorylation and gene expression in response to NMDA receptor activation. The phosphorylation of the transcription factor CREB, the expression of the immediate early gene c-fos, and the induction of a transfected reporter gene under the transcriptional control of CREB after stimulation of ionotropic glutamate receptors were investigated. Neither AMPA/kainate receptors nor NMDA receptors are able to independently stimulate a second messenger pathway that leads to CREB phosphorylation or c-fos gene expression. Instead, a consecutive pathway from AMPA/kainate receptors to NMDA receptors and from NMDA receptors to L-type Ca2+ channels is seen. AMPA/kainate receptors are involved in relieving the Mg2+ block of NMDA receptors, and NMDA receptors trigger the opening of L-type Ca2+ channels. The second messenger pathway that activates CREB phosphorylation and c-fos gene expression is likely activated by Ca2+ entry through L-type Ca2+ channels. It is concluded that in primary striatal neurons glutamate-mediated signal transduction is dependent on functional L-type Ca2+ channels (Rajadhyaksha, 1999).

AMPA/kainate receptor channels open after interaction with glutamate and permit Na+ entry at the synapse. The resulting local depolarization removes the Mg2+ block of the NMDA receptor, which permits the NMDA receptor to respond to extracellular glutamate and glycine. Opening of the NMDA receptor channel causes Na+ and Ca2+ influx. Unlike the AMPA/kainate receptor channel that desensitizes rapidly, NMDA receptor channels have long opening times. Therefore, NMDA receptors can trigger the opening of L-type Ca2+ channels that open during strong depolarization. The activation of L-type Ca2+ channels promotes Ca2+ entry along the dendrites and at the cell body. Second messengers activated by Ca2+ translocate to the nucleus and phosphorylate CREB. The results presented in this paper suggest an important role for L-type Ca2+ channels in neuroplasticity of the striatum and confirm previous reports about the involvement of L-type Ca2+ channels in NMDA-mediated plasticity and toxicity. Under the experimental conditions described in this study, NMDA receptors initiate a signal transduction pathway but do not initiate a significant intraneuronal second messenger pathway, either alone or together with AMPA/kainate receptors. Depolarization of L-type Ca2+ channels plays a crucial role in the activation of an intraneuronal second messenger pathway (Rajadhyaksha, 1999).

Although the supportive role of AMPA/kainate receptors for NMDA receptors is in agreement with previous findings in hippocampal culture, other findings differ. In hippocampal cultures NMDA receptors and L-type Ca2+ channels seem to contribute to independent, parallel pathways rather than to the same pathway. Like in hippocampal cultures, L-type Ca2+ channels in the striatum activate the CRE and function independently of NMDA receptors. But although a direct pathway from NMDA receptors to the SRE in the striatum cannot be excluded, this pathway in itself is not enough to mediate c-fos gene expression. This difference may be attributed to intrinsic differences between both types of neurons or to the different neurotransmitters released in either culture. Hippocampal neurons are mostly glutamatergic and express very high levels of glutamate receptors. Striatal cultures are primarily GABAergic and express much lower levels of glutamate receptors. Because neurons in culture synapse onto each other, hippocampal neurons excite each other after activation, whereas GABA in striatal neurons, dependent on the level of maturity, may be excitatory or inhibitory. To avoid trans-synaptic effects in hippocampal cultures, Na+ channels are often blocked with TTX. Thus, there are fundamental differences in glutamate-mediated gene expression in neurons of both brain areas (Rajadhyaksha, 1999).

Redistribution of postsynaptic AMPA-subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. AMPA receptors undergo clathrin-dependent endocytosis, which is accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreases AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation (LFS) induced homosynaptic CA1 LTD are found to be mutually occlusive and are both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS (Man, 2000).

Endocytosis of postsynaptic AMPA receptors may not be limited to homosynaptic CA1 LTD: insulin/IGF-I also produces a rapid and long-term depression of AMPA responses mediated by postsynaptic clathrin-dependent endocytosis in cultured cerebellar neurons. The insulin/IGF-I-induced depression of AMPA currents occludes cerebellar LTD, which in turn can be blocked by the inhibition of postsynaptic clathrin-dependent endocytosis. Additionally, a rapid, activity-dependent reduction of postsynaptic AMPA receptors takes place in a culture model of LTD induced by field stimulation. Taken together, these data suggest that rapid, clathrin-dependent removal of postsynaptic AMPA receptors may be a common final step in the expression of certain forms of LTD (Man, 2000 and references therein).

How is the clathrin-dependent endocytosis of AMPA receptors stimulated by LTD-inducing protocols? Since neurons contain and are able to release insulin in an activity-dependent manner, and since insulin receptors are concentrated in the postsynaptic density, one mechanism may involve the release of insulin presynaptically in response to LFS during LTD induction. Insulin may in turn activate its postsynaptic neuronal receptors to facilitate clathrin-dependent endocytosis of AMPA receptors. However, postsynaptic injection of the insulin receptor-neutralizing antibody, while blocking insulin-induced depression of AMPA EPSCs, has little effect on either hippocampal homosynaptic LTD or cerebellar LTD. These results suggest that insulin is not itself directly involved in mediating the expression of these forms of LTD but rather that insulin and LTD-inducing stimuli may converge to cause AMPA receptor endocytosis. It is likely that multiple signal transduction pathways exist for the regulation of AMPA receptor trafficking, and the elucidation of these pathways will provide further insight into the molecular mechanisms of synaptic plasticity and may ultimately provide mechanistic clues for the role of insulin in learning and memory (Man, 2000 and references therein).

Phosphorylation of the glutamate receptor is an important mechanism of synaptic plasticity. The C terminus of GluR2 of the AMPA receptor is phosphorylated by protein kinase C and serine-880 is the major phosphorylation site. This phosphorylation also occurs in human embryonic kidney (HEK) cells by addition of 12-O-tetradecanoylphorbol 13-acetate. Immunoprecipitation experiments reveal that the phosphorylation of serine-880 in GluR2 drastically reduces the affinity for glutamate receptor-interacting protein (GRIP), a synaptic PDZ domain-containing protein, in vitro and in HEK cells. This result suggests that modulation of serine-880 phosphorylation in GluR2 controls the clustering of AMPA receptors at excitatory synapses and consequently contributes to synaptic plasticity (Matsuda, 1999).

Cerebellar long-term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Using cultured Purkinje cells, it has been shown that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C-terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD (Matsuda, 2000).

Experience-dependent regulation of synaptic strength has been suggested as a physiological mechanism by which memory storage occurs in the brain. Although modifications in postsynaptic glutamate receptor levels have long been hypothesized to be a molecular basis for long-lasting regulation of synaptic strength, direct evidence obtained in the intact brain has been lacking. In the adult brain in vivo, synaptic glutamate receptor trafficking is bidirectionally, and reversibly, modified by NMDA receptor-dependent synaptic plasticity and changes in glutamate receptor protein levels accurately predict changes in synaptic strength. These findings support the idea that memories can be encoded by the precise experience-dependent assignment of glutamate receptors to synapses in the brain (Heynen, 2000).

LTP in vivo is associated with the delivery of glutamate receptor proteins to CA1 synapses, while LTD is associated with their removal. Like LTP and LTD, the changes in glutamate receptors depend on NMDAR activation during conditioning stimulation and are reversible. Although LTP results in an increase in synaptoneurosomal glutamate receptor protein levels, while LTD results in a decrease, these changes are not perfectly symmetric. LTP de novo correlates with an increase in AMPAR protein in CA1 synaptoneurosomes without a detectable change in NMDAR protein levels, while LTD de novo correlates with a decrease in both AMPAR and NMDAR protein in this biochemical fraction. These data demonstrate, for the first time, that a bidirectional redistribution of glutamate receptors accompanies bidirectional synaptic plasticity in the adult hippocampus in vivo (Heynen. 2000).

Activity-driven delivery of AMPA receptors is proposed to mediate glutamatergic synaptic plasticity, both during development and learning. In hippocampal CA1 principal neurons, such trafficking is primarily mediated by the abundant GluR-A subunit. A study of GluR-B_long, a C-terminal splice variant of the GluR-B subunit, is reported. GluR-B_long synaptic delivery is regulated by two forms of activity. Spontaneous synaptic activity-driven GluR-B_long transport maintains one-third of the steady-state AMPA receptor-mediated responses, while GluR-B_long delivery following the induction of LTP is responsible for approximately 50% of the resulting potentiation at the hippocampal CA3 to CA1 synapses at the time of GluR-B_long peak expression -- the second postnatal week. Trafficking of GluR-B_long-containing receptors thus mediates a GluR-A-independent form of glutamatergic synaptic plasticity in the juvenile hippocampus (Kolleker, 2003).

Synaptic plasticity involves protein phosphorylation cascades that alter the density of AMPA-type glutamate receptors at excitatory synapses; however, the crucial phosphorylated substrates remain uncertain. The AMPA receptor-associated protein stargazin has been shown to be quantitatively phosphorylated, and stargazin phosphorylation promotes synaptic trafficking of AMPA receptors. Synaptic NMDA receptor activity can induce both stargazin phosphorylation, via activation of CaMKII and PKC, and stargazin dephosphorylation, by activation of PP1 downstream of PP2B. At hippocampal synapses, long-term potentiation and long-term depression require stargazin phosphorylation and dephosphorylation, respectively. These results establish stargazin as a critical substrate in the bidirectional control of synaptic strength, which is thought to underlie aspects of learning and memory (Tomita, 2005).

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. This study reports that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses (Zhu, 2005).

Activity-dependent synaptic delivery of GluR1-, GluR2L-, and GluR4-containing AMPA receptors (-Rs) and removal of GluR2-containing AMPA-Rs mediate synaptic potentiation and depression, respectively. The obvious puzzle is how synapses maintain the capacity for bidirectional plasticity if different AMPA-Rs are utilized for potentiation and depression. This study shows that synaptic AMPA-R exchange is essential for maintaining the capacity for bidirectional plasticity. The exchange process consists of activity-independent synaptic removal of GluR1-, GluR2L-, or GluR4-containing AMPA-Rs and refilling with GluR2-containing AMPA-Rs at hippocampal and cortical synapses in vitro and in intact brains. In GluR1 and GluR2 knockout mice, initiation or completion of synaptic AMPA-R exchange is compromised, respectively. The complementary AMPA-R removal and refilling events in the exchange process ultimately maintain synaptic strength unchanged, but their long rate time constants (15-18 hr) render transmission temporarily depressed in the middle of the exchange. These results suggest that the previously hypothesized 'slot' proteins, rather than AMPA-Rs, code and maintain transmission efficacy at central synapses (McCormack, 2006).

How synaptic AMPA-R exchange maintains transmission efficacy is unclear. It is possible that during exchange, GluR2-containing AMPA-Rs get into synapses and make a one-to-one replacement of synaptic GluR1-, GluR2L-, or GluR4-containing AMPA-Rs. It is also possible that synaptic delivery of GluR1-, GluR2L-, and GluR4-containing AMPA-Rs brings with them 'slot' proteins, which allow GluR2-containing AMPA-Rs to refill the empty 'slots' after GluR1-, GluR2L-, and GluR4-containing AMPA-Rs leave synapses. The temporarily depressed AMPA responses during synaptic AMPA-R exchange indicate that GluR1-, GluR2L-, and GluR4-contaning AMPA-Rs leave synapses before GluR2-containing AMPA-Rs fill in. This view is further supported by findings that synaptic refilling of GluR2-containing AMPA-Rs after removal of AMPA-Rs with long cytoplasmic termini are required for completing exchange and maintaining transmission unaltered after exchange. Because the ultimate transmission strength does not change after the exchange, synaptic efficacy must be 'memorized' by molecule(s) other than AMPA-Rs, in particular when AMPA-R-mediated transmission is temporarily depressed. The results thus provide experimental evidence supporting the 'slot' theory: 'slot' proteins, instead of AMPA-Rs, code and maintain transmission efficacy. The remaining puzzle is what are the 'slot' proteins. Proteomic analysis and functional characterization of proteins binding to cytoplasmic termini of all AMPA-R subunits promise to reveal their identity (McCormack, 2006).

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. This study shows that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum (Steinberg, 2006).

Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. GSK3β mediates an interaction between two major forms of synaptic plasticity in the brain, NMDA receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3β inhibitors block the induction of LTD. Furthermore, the activity of GSK3β is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3β activity. This regulation of GSK3β during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. It is concluded that the regulation of GSK3β activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information (Peineau, 2007).

NMDA receptor-dependent LTD is due to the internalization of AMPA receptors and involves protein interactions directly associated with the AMPA receptor subunits, particularly GluR2. It was reasoned that GSK3β might form a complex with AMPA receptors, and thus attempts were made to investigate this by probing for an association of native GSK3β with AMPA receptors in the CA1 area of hippocampal slices. A specific antibody against GSK3β was able to coimmunoprecipitate the GluR1 and GluR2 AMPA receptor subunits, and conversely, immunoprecipitation of AMPA receptors produced coimmunoprecipitation of GSK3β. To determine the functional status of AMPA receptor-associated GSK3β, AMPA receptors were immunoprecipitated, using antibodies against either GluR1 or GluR2, and then assayed for kinase activity. GSK3β activity was readily detected in both GluR1 and GluR2 immunoprecipitates relative to the background IgG control, demonstrating that endogenous GSK3β associates with native AMPA receptors in the brain, and that the bound GSK3β is functionally active. This association of GSK3β with AMPA receptors suggests a compartmentalization of this enzyme for the efficient regulation of AMPA receptors during LTD (Peineau, 2007).

It was asked whether the GSK3β activity that is associated with AMPA receptors could be regulated. Previous work has shown that transient exposure of cultured neurons to a solution containing sucrose plus glycine leads to an NMDA receptor-dependent insertion of AMPA receptors into the plasma membrane. Interestingly, this effect is associated with an increase in AMPA receptor-associated PI3K activity. Since PI3K is an upstream regulator of GSK3β, whether this treatment also affected the AMPA receptor-associated GSK3β enzyme activity was investigated. Neurons were treated with sucrose (200 mM) plus glycine (100 µM) for 2 min, and this led to the insertion of AMPA receptors into the plasma membrane as determined approximately 15 min later using surface biotinylation assays. This chemically induced AMPA receptor insertion was associated with a decrease in AMPA receptor-associated GSK3β activity (Peineau, 2007).

This study identified a form of regulation of synaptic plasticity in which the transient synaptic activation of NMDA receptors, as occurs during LTP, leads to inhibition of LTD. This regulation is very powerful since LTD is fully inhibited immediately following the conditioning stimulus and the effect lasts for approximately 1 hr. Also some of the signaling pathways responsible for this potent regulation of synaptic plasticity have been identified. GSK3β activity is an absolute requirement for the induction of LTD and the conditioning stimulus inhibits its activity via activation of the PI3K-Akt pathway. Finally, there is a correlation between the phosphorylation state of GSK3β ser9 and whether NMDA receptor activation leads to the induction or inhibition of LTD (Peineau, 2007).

GSK3β is an unusual kinase that has been implicated in many diseases. However, very little is known about its normal function in the nervous system. It is important during early development and it has been shown to play a key role in cell polarity and in the growth of neuromuscular junctions. Recently, it has been shown that GSK3β is important for determining neuronal polarity during the development of hippocampal neurons. However, though GSK3β is also highly expressed in the mature brain, its function in the nervous system has, hitherto, been largely unexplored. In the nucleus of hippocampal neurons, GSK3β is involved in the regulation of gene transcription by promoting the nuclear export of the transcription factor NF-ATc4. In addition, it has been shown that overexpression of GSK3β impairs spatial learning, though the mechanism underlying this effect is unknown. This study shows that in 2-week-old rats, an age at which the expression of GSK3β is near its peak, GSK3β activity is essential for NMDA receptor-dependent LTD in the hippocampus. This form of LTD is widespread throughout the brain and has been strongly implicated in development and learning and memory. Therefore, this novel GSK3β-dependent mechanism may be of general significance in regulating the interaction between LTP and LTD throughout the brain (Peineau, 2007).

GSK3β, unlike most enzymes, possesses high basal level constitutive activity and can be bidirectionally regulated to either further increase or decrease its activity. During LTD there is additional activation of GSK3β, probably via dephosphorylation of ser9. This effect is prevented by an inhibitor of PP1/PP2A. This suggests that the activation of PP1, which is known to occur during LTD, is responsible for the activation of GSK3β, via its dephosphorylation of ser9. LTD is associated with inhibition of Akt, probably also via the activation of PP1. These data suggest that GSK3β activity is increased during LTD because the phosphatase concomitantly inhibits Akt and directly dephosphorylates ser9 of GSK3β (Peineau, 2007).

Interestingly, the alteration in the phosphorylation status of GSK3β persists beyond the delivery of low-frequency stimulation (LFS), and lithium completely blocks LTD when applied after the delivery of LFS. These data suggest that GSK3β is required for the LTD process beyond the initial induction phase. Further studies are required to determine the full time course of the involvement of GSK3β in LTD (Peineau, 2007).

GSK3β has several upstream regulators and numerous downstream targets. In the present study, two of its upstream regulators have been identified. During LTD, GSK3β is activated via an okadaic acid-sensitive protein phosphatase, which is probably PP1. During LTP, GSK3β is inhibited via the PI3K-Akt pathway. Since GSK3β is such a ubiquitous kinase, it needs mechanisms to localize its access to its substrates. This is achieved in part via direct interactions with other proteins to form complexes. For example, in the canonical Wnt pathway, GSK3β binding proteins control access of β-catenin. It seems likely that the association between GSK3β and AMPA receptors serves to localize the kinase close to substrates that are involved in the trafficking of these receptors during synaptic plasticity. Further studies are required to establish the mechanism of this interaction as well as the downstream pathways mediated by GSK3β in the regulation of LTD (Peineau, 2007).

The finding that the synaptic activation of NMDA receptors during LTP inhibits NMDA receptor-dependent LTD raises an intriguing issue: what determines whether the synaptic activation of NMDA receptors leads to the induction or inhibition of LTD? Evidence is presented that the phosphorylation state of ser9 of GSK3β is a critical determinant. Thus, during LTP, activation of the PI3K-Akt pathway results in phosphorylation of GSK3β, and hence inhibition of its activity. In contrast, during LTD, activation of PP1 results in inhibition of Akt and the dephosphorylation of GSK3β at ser9, and this leads to an increase in the enzyme's activity. The activation of PI3K-Akt and inhibition of PP1 during LTP, but inhibition of Akt during LTD as well as the selective activation of PP1 during LTD, can be explained by the differences in the magnitude and spatiotemporal properties of the Ca2+ rise associated with the synaptic activation of NMDA receptors during these two forms of synaptic plasticity (Peineau, 2007).

Previous work has described other ways in which synaptic plasticity can be powerfully influenced by the prior history of synaptic activity. However, the mechanisms involved in these forms of metaplasticity are not known. Why synapses need such regulatory mechanisms is a matter of conjecture. One intriguing possible role for the regulation described in this study is to stabilize a synaptic modification over the short term by protecting synapses from the effects of additional NMDA receptor-dependent plasticity until the information can be either consolidated or erased by NMDA receptor-independent mechanisms (Peineau, 2007).

The regulation of synaptic plasticity is further complicated by the involvement of mGluRs, which are involved in depotentiation, LTD of baseline transmission, heterosynaptic LTD, and metaplasticity. So that focus could be placed on interactions between the NMDA receptor-dependent forms of synaptic plasticity, the additional complication of mGluR-dependent synaptic plasticity were eliminated by using the broad spectrum mGluR antagonist LY341495 and by employing stimulus protocols optimized for NMDA receptor-dependent synaptic plasticity. However, given that PI3K has been implicated in a chemically induced form of mGluR-dependent LTD and heterosynaptic LTD, it will be interesting to determine whether GSK3β is also involved in these forms of synaptic plasticity. One possibility is that the PI3K-Akt-GSK3β pathway serves to inhibit NMDA receptor-dependent LTD both homosynaptically following the induction of LTP and heterosynaptically following the induction of LTD (Peineau, 2007).

The finding that in the normal brain activation of GSK3β is essential for NMDA receptor-dependent LTD, and that its activity can be regulated by LTP, may offer clues to the pathological role of this enzyme in neurological disorders. For example, the primary therapeutic action of lithium in bipolar disorders may be via inhibition of GSK3β. Indeed, specific inhibition of GSK3β has recently been shown to produce antidepressive-like activity in vivo. Overactivity of GSK3β may, therefore, lead to this mood disorder by affecting the balance and interplay between NMDA receptor-dependent LTP and LTD (Peineau, 2007).

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. Evidence is presented that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibits NMDA-induced AMPA receptor internalization and blocks the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295 (Kim, 2007).

Glutamate receptors and paired-associated learning

Paired-associate learning is often used to examine episodic memory in humans. Animal models include the recall of foodcache locations by scrub jays and sequential memory. This study reports a model in which rats encode, during successive sample trials, two paired associates (flavors of food and their spatial locations) and display better-than-chance recall of one item when cued by the other. In a first study, pairings of a particular foodstuff and its location were never repeated, so ensuring unique 'what-where' attributes. Blocking NMDA receptors in the hippocampus -- crucial for the induction of certain forms of activity-dependent synaptic plasticity -- impairs memory encoding but has no effect on recall. Inactivating hippocampal neural activity by blocking AMPA receptors impairs both encoding and recall. In a second study, two paired associates were trained repeatedly over 8 weeks in new pairs, but blocking of hippocampal AMPA receptors does not affect their recall. Thus it is concluded that unique what-where paired associates depend on encoding and retrieval within a hippocampal memory space, with consolidation of the memory traces representing repeated paired associates in circuits elsewhere (Day, 2003).

AMPA receptors and direction-selective and spatial phase-selective responses in the visual cortex

Cells in the superficial layers of primary visual cortex (area 17) are distinguished by feedforward input from thalamic-recipient layers and by massive recurrent excitatory connections between neighboring cells. The connections use glutamate as transmitter, and the postsynaptic cells contain both NMDA and AMPA receptors. The possible role of these receptor types in generating emergent responses of neurons in the superficial cortical layers is unknown. NMDA and AMPA receptors are both involved in the generation of direction-selective responses in layer 2/3 cells of area 17 in cats. NMDA receptors contribute prominently to responses in the preferred direction, and their contribution to responses in the nonpreferred direction is reduced significantly by GABAergic inhibition. AMPA receptors decrease spatial phase-selective simple cell responses and generate phase-invariant complex cell responses (Rivadulla, 2001).

By combining extracellular recording and iontophoresis of receptor blockers, the following results have been demonstrated: (1) Blocking AMPA receptors removes a proportionately larger component from nonpreferred compared with preferred responses and increases direction selectivity. The remaining responses are mediated by NMDA receptors and are overwhelmingly in the preferred direction. (2) Blocking NMDA receptors removes proportional components from preferred and nonpreferred responses and preserves directional selectivity. Because the remaining responses are mediated by AMPA receptors, these receptors are sufficient for direction selectivity. (3) Blocking inhibition preferentially enhances the contribution of NMDA receptors to nonpreferred responses and reduces direction selectivity. Thus, inhibition contributes to direction selectivity by reducing NMDA responses in the nonpreferred direction. (4) Blocking AMPA receptors increases the modulation of complex cell responses by a drifting grating stimulus. Thus, AMPA receptors decrease the selectivity of complex cells for spatial phase or the spatial location of visual stimuli. (5) Blocking NMDA receptors or inhibition has little effect on the temporal modulation of simple or complex cell responses. Together, these results allow for the proposal specific roles for NMDA and AMPA receptors in direction selectivity in the superficial layers of area 17 and in the generation of phase selectivity by simple and complex cells in these layers (Rivadulla, 2001).

Direction selectivity first appears in simple cells of layer 4 in area 17, where NMDA receptors are not present in significant proportions and contribute little to visual responses. The mechanism(s) by which direction selectivity is generated and whether the mechanism is similar in various cortical layers remain unresolved. One hypothesis is that inhibition reduces the response in the nonpreferred direction. The hypothesis is supported by pharmacological studies in cat and monkey, demonstrating that blockade of inhibition in cortical cells induces a loss of selectivity to the direction of stimulus motion. An alternative hypothesis is that there is enhancement of excitation in the preferred direction. It has been shown that simple cells in area 17 have asymmetries in the time course of the response evoked from different positions of the receptive field. Linear summation of these asymmetries allows one to predict the direction preference of the cell but also leads to an overestimation of the response in the nonpreferred direction. Recurrent excitation has been proposed as a nonlinear mechanism by which responses can be increased in the preferred direction. Recently, it has been postulated that inhibition can sculpt the spatiotemporal profile of the receptive field, accentuating spatiotemporal asymmetry and increasing direction selectivity, particularly in layer 4 (Rivadulla, 2001 and references therein).

A fundamental difference between layers 2/3 and 4 is the presence in supragranular layers of NMDA receptors, where they have been shown to participate in transmission in vivo and in vitro. AMPA and NMDA receptors both contribute to direction selectivity in supragranular layers. AMPA receptors are sufficient for generating direction selectivity, either because inputs to the superficial layers conveyed by AMPA receptors are already biased for direction or because feedforward and recurrent connections mediated by AMPA receptors generate direction selectivity within these layers. NMDA receptors by themselves can generate highly direction-selective responses, by summing and/or amplifying responses to the preferred stimulus while contributing less to nonpreferred responses because of close GABAergic control. One possibility is that NMDA receptor activation is possible only with enough excitation in the preferred direction. However, a comparison of the nonpreferred and spontaneous responses that remain after application of AMPA receptor inhibitor CNQX (spontaneous activity in this population of cells is reduced on average by only 28% under CNQX, whereas nonpreferred responses are reduced by 88%) suggests that the reduced contribution of NMDA receptors to nonpreferred responses is likely mediated by active inhibition rather than simply being a function of overall response magnitude: spontaneous activity occurs under less inhibition than nonpreferred responses and remains significantly greater after AMPA receptor blockade (Rivadulla, 2001).

Two lines of evidence indicate that GABAergic inhibition regulates the reduced contribution of NMDA receptors to nonpreferred responses. (1) Blocking inhibition by application of bicuculline decreases the direction selectivity of cells, but this effect is reversed by simultaneous application of APV, indicating that release of inhibition facilitates NMDA responses in the nonpreferred direction. (2) Blockade of inhibition concurrent with AMPA receptor blockade by CNQX reduces direction selectivity. Bicuculline preferentially increases nonpreferred responses, leaving a higher contribution of NMDA responses in the nonpreferred direction (Rivadulla, 2001).

These data confirm and extend the findings that NMDA inhibitor APV causes a proportional reduction in responses of area 17 cells to optimally oriented moving bars as stimulus contrast is increased, whereas application of NMDA increases responses by a proportional amount and quisqualate increases responses by an absolute amount at all contrasts. Importantly, responses in different directions (as also simple and complex cell responses) were studied with APV and CNQX and the modulation of NMDA and AMPA responses by inhibition. NMDA-mediated responses in the nonpreferred direction are reduced nonlinearly by inhibition. Furthermore, the contribution of AMPA receptors to complex cell responses is much more than addition of a constant response component; rather, there is a nonlinear change in the temporal modulation of the response. These observations argue for specific circuits that engage inhibition for generating direction-selective responses and AMPA receptors for generating complex cell responses (Rivadulla, 2001).

The possibility that inhibition regulates NMDA-mediated activity is consistent with other lines of evidence in area 17. The relationship between GABAergic inhibition and NMDA function is probably related to the voltage dependence of NMDA receptors; the binding of extracellular Mg2+ to the channel pore is highly dependent on membrane potential, and changes in the latter could significantly modulate NMDA receptor-mediated activity. The likely source of inhibition is GABAergic interneurons located within layer 2/3 itself (Rivadulla, 2001).

One caveat is that the iontophoresis technique does not allow definitive conclusions about whether all receptors on a cell are affected or whether other cells (either excitatory or inhibitory) in the vicinity could be modifying the responses of the recorded cell. However, the temporal effects of drug application were studied in the first and the second half of the iontophoresis period in several cells and found to be similar. Furthermore, the effect of iontophoresis does not change with ejection time, indicating that most of the affected receptors are in the volume covered by the antagonist since the start of iontophoresis (Rivadulla, 2001).

In addition to examining the role of AMPA and NMDA receptors in direction selectivity, an examination was made of their role in generating simple and complex responses in the supragranular layers by analyzing the temporal pattern of response of cells when they were stimulated with drifting gratings. CNQX caused a dramatic change in complex cell responses, causing them to increase their temporal modulation and respond in a manner similar to that of simple cells. APV does not affect the response modulation of complex cells, and the modulation of simple cell responses is unaffected by CNQX or APV (Rivadulla, 2001).

Recently, it has been proposed that complex cell responses arise as a consequence of decreasing the phase selectivity of simple cell responses by recurrent intracortical connections. The model predicts that a decrease in intracortical excitation should cause complex cells to respond like simple cells. If AMPA receptors primarily mediate short-range intracortical excitation, the results presented here agree strongly with this prediction, demonstrating that during blockade of AMPA receptors complex cells behave as simple cells when stimulated with drifting gratings (Rivadulla, 2001).

It is proposed that AMPA and NMDA receptors in layer 2/3 have different spatial distributions on cells, with both present on the same cell but in different proportions at different inputs. Both receptors mediate feedforward connections, and these afferents provide the necessary input for direction selectivity in layer 2/3. The data are consistent with spatiotemporal asymmetry and enhancement of excitation in feedforward pathways as crucial for direction selectivity in layer 2/3, with a prominent role for NMDA receptors in generating the preferred response and a role for GABAergic inhibition in reducing the nonpreferred response. In contrast to feedforward connections, local recurrent connections are mainly mediated by AMPA receptors (with a possible small contribution from NMDA receptors), and they are responsible for smearing the phase selectivity of simple cells to create phase-invariant complex cell responses (Rivadulla, 2001).

The suggestion that short-range excitation between cortical cells is mediated primarily by AMPA receptors is consistent with the fact that fast EPSCs that are evoked in supragranular layer cells in area 17 by adjacent intralaminar stimulation are not APV sensitive. In somatosensory cortex, intracellular recording of unitary EPSCs in layer 4 and the supragranular cortex indicates that both thalamocortical and intracortical EPSCs are mediated by AMPA receptors and have similar characteristics. In slices of area 17, white matter stimulation evokes EPSPs in the supragranular layers that have NMDA- and AMPA-mediated components. Because of feedforward and local recurrent connections, short-latency responses are primarily AMPA mediated, whereas long-latency responses, because of horizontal connections, have significant NMDA components. Furthermore, long-range horizontal inputs to layer 2/3 cells in area 17 can sum nonlinearly with feedforward or short-range inputs, indicative of NMDA receptor involvement in the long-range connections. Thus, it is likely that there is even finer spatial segregation of glutamate receptors associated with specific inputs on layer 2/3 cells. Together with the modulation of responses (particularly those mediated by NMDA receptors) by inhibition, the specific relationship between receptor types and anatomical connections provides a rich substrate for dynamic control of emergent responses in the cortex (Rivadulla, 2001).

AMPA receptors and spinal synaptic plasticity and inflammatory pain

Ca2+-permeable AMPA receptors are densely expressed in the spinal dorsal horn, but their functional significance in pain processing is not understood. By disrupting the genes encoding GluR-A or GluR-B, mice were generated exhibiting increased or decreased numbers of Ca2+-permeable AMPA receptors, respectively. AMPA receptors are critical determinants of nociceptive plasticity and inflammatory pain. A reduction in the number of Ca2+-permeable AMPA receptors and density of AMPA channel currents in spinal neurons of GluR-A-deficient mice is accompanied by a loss of nociceptive plasticity in vitro and a reduction in acute inflammatory hyperalgesia in vivo. In contrast, an increase in spinal Ca2+-permeable AMPA receptors in GluR-B-deficient mice facilitates nociceptive plasticity and enhances long-lasting inflammatory hyperalgesia. Thus, AMPA receptors are not mere determinants of fast synaptic transmission underlying basal pain sensitivity, but are critically involved in activity-dependent changes in synaptic processing of nociceptive inputs (Hartmann, 2005).

Glutamate receptors and cocaine addiction

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. This study generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. In midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior (Engblom, 2008).

A single exposure to drugs of abuse produces an NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA receptor (AMPAR) currents in DA neurons; however, the importance of LTP for various aspects of drug addiction is unclear. To test the role of NMDAR-dependent plasticity in addictive behavior, functional NMDAR signaling was genetically inactivated exclusively in DA neurons (KO mice). Inactivation of NMDARs results in increased AMPAR-mediated transmission that is indistinguishable from the increases associated with a single cocaine exposure, yet locomotor responses to multiple drugs of abuse were unaltered in the KO mice. The initial phase of locomotor sensitization to cocaine is intact; however, the delayed sensitization that occurs with prolonged cocaine withdrawal did not occur. Conditioned behavioral responses for cocaine-testing environment were also absent in the KO mice. These findings provide evidence for a role of NMDAR signaling in DA neurons for specific behavioral modifications associated with drug seeking behaviors (Zweifel, 2008).

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