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AMPA-receptor trafficking plays a central role in excitatory plasticity, especially during development. Changes in the number of AMPA receptors and time spent at the synaptic surface are important factors of plasticity that directly affect long-term potentiation (LTP), long-term depression (LTD), synaptic scaling, and the excitatory-inhibitory (E/I) balance in the developing cortex. Experience-dependent changes in synaptic strength in visual cortex (V1) use a molecularly distinct AMPA trafficking pathway that includes the GluA2 subunit. We studied developmental changes in AMPA receptor trafficking proteins by quantifying expression of GluA2, pGluA2 (GluA2serine880), GRIP1, and PICK1 in rat visual and frontal cortex. We used Western Blot analysis of synaptoneurosome preparations of rat visual and frontal cortex from animals ranging in age from P0 to P105. GluA2 and pGluA2 followed different developmental trajectories in visual and frontal cortex, with a brief period of over expression in frontal cortex. The over expression of GluA2 and pGluA2 in immature frontal cortex raises the possibility that there may be a period of GluA2-dependent vulnerability in frontal cortex that is not found in V1. In contrast, GRIP1 and PICK1 had the same developmental trajectories and were expressed very early in development of both cortical areas. This suggests that the AMPA-interacting proteins are available to begin trafficking receptors as soon as GluA2-containing receptors are expressed. Finally, we used all four proteins to analyze the surface-to-internalization balance and found that this balance was roughly equal across both cortical regions, and throughout development. Our finding of an exquisite surface-to-internalization balance highlights that these AMPA receptor trafficking proteins function as a tightly controlled system in the developing cortex.

We have shown altered expression of gamma-aminobutyric acid A (GABAA) and gamma-aminobutyric acid B (GABAB) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3 GABAA subunits previously associated with autism (GABRα4; GABRα5; GABRβ1). Three GABA receptor subunits demonstrated mRNA and protein level concordance in superior frontal cortex (GABRα4, GABRα5, GABRβ1) and one demonstrated concordance in cerebellum (GABBR1). These results provide further evidence of impairment of GABAergic signaling in autism.

Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca2+-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left–right asymmetry of hippocampal CA3–CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD) area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supralinear (GluR1 AMPA) or inverse (NR2B NMDAR) relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.

Synaptic plasticity in the amygdala is essential for emotional learning. Fear conditioning, for example, depends on changes in excitatory transmission that occur following NMDA receptor activation and AMPA receptor modification in this region. The role of these and other glutamatergic mechanisms have been studied extensively in this circuit while relatively little is known about the contribution of inhibitory transmission. The current experiments addressed this issue by examining the role of the GABA(A) receptor subunit α1 in fear learning and plasticity. We first confirmed previous findings that the α1 subunit is highly expressed in the lateral nucleus of the amygdala. Consistent with this observation, genetic deletion of this subunit selectively enhanced plasticity in the lateral amygdala and increased auditory fear conditioning. Mice with selective deletion of α1 in excitatory cells did not exhibit enhanced learning. Finally, infusion of a α1 receptor antagonist into the lateral amygdala selectively impaired auditory fear learning. Together, these results suggest that inhibitory transmission mediated by α1-containing GABA(A) receptors plays a critical role in amygdala plasticity and fear learning.

Functional inhibitory synapses form in auditory cortex well before the onset of normal hearing. However, their properties change dramatically during normal development, and many of these maturational events are delayed by hearing loss. Here, we review recent findings on the developmental plasticity of inhibitory synapse strength, kinetics, and GABAA receptor localization in auditory cortex. Although hearing loss generally leads to a reduction of inhibitory strength, this depends on the type of presynaptic interneuron. Furthermore, plasticity of inhibitory synapses also depends on the postsynaptic target. Hearing loss leads reduced GABAA receptor localization to the membrane of excitatory, but not inhibitory neurons. A reduction in normal activity in development can also affect the use-dependent plasticity of inhibitory synapses. Even moderate hearing loss can disrupt inhibitory short- and long-term synaptic plasticity. Thus, the cortex did not compensate for the loss of inhibition in the brainstem, but rather exacerbated the response to hearing loss by further reducing inhibitory drive. Together, these results demonstrate that inhibitory synapses are exceptionally dynamic during development, and deafness-induced perturbation of inhibitory properties may have a profound impact on auditory processing.

Functional maturation of visual cortex is linked with dynamic changes in synaptic expression of GABAergic mechanisms. These include setting the excitation–inhibition balance required for experience-dependent plasticity, as well as, intracortical inhibition underlying development and aging of receptive field properties. Animal studies have shown that there is developmental regulation of GABAergic mechanisms in visual cortex. In this study, we show for the first time how these mechanisms develop in the human visual cortex across the lifespan. We used Western blot analysis of postmortem tissue from human primary visual cortex (n = 30, range: 20 days to 80 years) to quantify expression of eight pre- and post-synaptic GABAergic markers. We quantified the inhibitory modulating cannabinoid receptor (CB1), GABA vesicular transporter (VGAT), GABA synthesizing enzymes (GAD65/GAD67), GABAA receptor anchoring protein (Gephyrin), and GABAA receptor subunits (GABAAα1, GABAAα2, GABAAα3). We found a complex pattern of different developmental trajectories, many of which were prolonged and continued well into the teen, young adult, and even older adult years. These included a monotonic increase or decrease (GABAAα1, GABAAα2), a biphasic increase then decrease (GAD65, Gephyrin), or multiple increases and decreases (VGAT, CB1) across the lifespan. Comparing the balances between the pre- and post-synaptic markers we found three main transition stages (early childhood, early teen years, aging) when there were rapid switches in the composition of the GABAergic signaling system, indicating that functioning of the GABAergic system must change as the visual cortex develops and ages. Furthermore, these results provide key information for translating therapies developed in animal models into effective treatments for amblyopia in humans.

Orientation selectivity (OS) of visual cortical neurons is progressively sharpened during development. However, synaptic circuit mechanisms underlying the OS sharpening remain unclear. In the current study, in vivo whole-cell voltage-clamp recordings from layer 4 excitatory neurons in the developing mouse primary visual cortex revealed changes of orientation tuning profiles of their excitatory and inhibitory inputs during a post eye-opening period when OS of their spiking responses becomes sharpened. Besides a parallel strengthening of excitation and inhibition during this developmental period, the orientation tuning of excitatory inputs keeps relatively constant, whereas the tuning of inhibitory inputs is broadened, and becomes significantly broader than that of excitatory inputs. Neuron modelling and dynamic-clamp recording demonstrated that this developmental broadening of the inhibitory tuning is sufficient for sharpening OS. Depriving visual experience by dark rearing impedes the normal developmental strengthening of excitation, but a similar broadening of inhibitory tuning, likely caused by a non-selective strengthening of inhibitory connections, results in the apparently normal OS sharpening in excitatory neurons. Our results thus provide the first demonstration that an inhibitory synaptic mechanism can primarily mediate the functional refinement of cortical neurons.

In the present study, we used a mouse model of chronic intermittent ethanol (CIE) exposure to examine how CIE alters the plasticity of the medial prefrontal cortex (mPFC). In acute slices obtained either immediately or 1-week after the last episode of alcohol exposure, voltage-clamp recording of excitatory post-synaptic currents (EPSCs) in mPFC layer V pyramidal neurons revealed that CIE exposure resulted in an increase in the NMDA/AMPA current ratio. This increase appeared to result from a selective increase in the NMDA component of the EPSC. Consistent with this, Western blot analysis of the postsynaptic density fraction showed that while there was no change in expression of the AMPA GluR1 subunit, NMDA NR1 and NRB subunits were significantly increased in CIE exposed mice when examined immediately after the last episode of alcohol exposure. Unexpectedly, this increase in NR1 and NR2B was no longer observed after 1-week of withdrawal in spite of a persistent increase in synaptic NMDA currents. Analysis of spines on the basal dendrites of layer V neurons revealed that while the total density of spines was not altered, there was a selective increase in the density of mushroom-type spines following CIE exposure. Examination of NMDA-receptor mediated spike-timing-dependent plasticity (STDP) showed that CIE exposure was associated with altered expression of long-term potentiation (LTP). Lastly, behavioral studies using an attentional set-shifting task that depends upon the mPFC for optimal performance revealed deficits in cognitive flexibility in CIE exposed mice when tested up to 1-week after the last episode of alcohol exposure. Taken together, these observations are consistent with those in human alcoholics showing protracted deficits in executive function, and suggest these deficits may be associated with alterations in synaptic plasticity in the mPFC.

In the cortex, homeostatic plasticity appears as a key process for maintaining neuronal networks activity in a functional range. This phenomenon depends on close interactions between excitatory and inhibitory circuits. We previously showed that application of a high-frequency of stimulation (HFS) protocol in layer 2/3 induces parallel potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons, leading to an unchanged excitation/inhibition (E/I) balance. These coordinated Long Term Potentiations (LTP) of excitation and inhibition correspond to homeostatic plasticity of the neuronal networks.

We showed here on the rat visual cortex, that the blockade (with gabazine, GBZ) or the over-activation (with 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP) of GABAA receptors enhanced the E/I balance and prevented the potentiation of excitatory and inhibitory inputs after an HFS protocol. These impairements of the GABAergic transmission led to a LTD-like (Long Term Depression) effect after an HFS protocol. We also observed that the blockade of inhibition reduced excitation (by 60 %) and conversely the blockade of excitation decreased inhibition (by 90 %). These results support the idea that inhibitory interneurons are critical for recurrent interactions underlying homeostatic plasticity in cortical networks.

Regulation of synaptic AMPA receptors (AMPARs) is one of the key elements that allow the nervous system to adapt to changes in the sensory environment as well as for memory formation. One way to regulate AMPAR function is by reversible changes in the phosphorylation of its subunits. We recently reported that phosphorylation of the AMPAR subunit GluA1 (or GluR1) on serine-845 (S845) is a pre-requisite step for sensory experience-dependent homeostatic synaptic plasticity in the visual cortex. In particular, increasing GluA1-S845 phosphorylation upregulated cell surface and synaptic AMPAR levels. Here we report that this is rather specific to the visual cortex, in that increasing GluA1-S845 phosphorylation in hippocampal slices only increase cell surface expression, but not synaptic AMPAR function. Our results suggest that depending on the brain region divergent mechanisms may exist to regulate synaptic AMPAR function with phosphorylation.

Experience-driven activity plays an essential role in the development of brain circuitry during critical periods of early postnatal life, a process that depends upon a dynamic balance between excitatory and inhibitory signals. Since general anesthetics are powerful pharmacological modulators of neuronal activity, an important question is whether and how these drugs can affect the development of synaptic networks. To address this issue, we examined here the impact of anesthetics on synapse growth and dynamics. We show that exposure of young rodents to anesthetics that either enhance GABAergic inhibition or block NMDA receptors rapidly induce a significant increase in dendritic spine density in the somatosensory cortex and hippocampus. This effect is developmentally regulated; it is transient but lasts for several days and is also reproduced by selective antagonists of excitatory receptors. Analyses of spine dynamics in hippocampal slice cultures reveals that this effect is mediated through an increased rate of protrusions formation, a better stabilization of newly formed spines, and leads to the formation of functional synapses. Altogether, these findings point to anesthesia as an important modulator of spine dynamics in the developing brain and suggest the existence of a homeostatic process regulating spine formation as a function of neural activity. Importantly, they also raise concern about the potential impact of these drugs on human practice, when applied during critical periods of development in infants.

In the cortex, NMDA receptors (NMDARs) play a critical role in the control of synaptic plasticity processes. We have previously shown in rat visual cortex that the application of a high frequency of stimulation (HFS) protocol used to induce long-term potentiation (LTP) in layer 2/3 leads to a parallel potentiation of excitatory and inhibitory inputs received by cortical layer 5 pyramidal neurons without changing the excitation/inhibition (E/I) balance of the pyramidal neuron, indicating a homeostatic control of this parameter.

We show here that the blockade of NMDARs of the neuronal network prevents the potentiation of excitatory and inhibitory inputs and this result opens to question the role of the NMDAR isoform involved in the induction of LTP, actually being strongly debated. In P18-P23 rat cortical slices, the blockade of synaptic NR2B-containing NMDARs prevents the induction of the potentiation induced by the HFS protocol, whereas the blockade of NR2A-containing NMDARs reduced the potentiation itself. In P29-P32 cortical slices, the specific activation of NR2A-containing receptors fully ensures the potentiation of excitatory and inhibitory inputs. These results constitute the first report of a functional shift in subunit composition of NMDARs during the critical period (P12-P36) which explains the relative contribution of both NR2B- and NR2A-containing NMDARs in synaptic plasticity processes. These effects of HFS protocol are mediated by the activation of synaptic NMDARs but our results also indicate that the homeostatic control of the E/I balance is independent of NMDARs activation and is due to specialized recurrent interactions between excitatory and inhibitory networks.

Kainate receptors mediate both direct excitatory and indirect modulatory actions in the CNS. We report here that kainate has both pre- and postsynaptic actions in layer II/III pyramidal neurons of rat prefrontal cortex. Application of low concentration of kainate (50-500 nM) increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) whereas higher concentrations (3 μM) caused a decrease. The frequency of spontaneous and miniature (action potential-independent) EPSCs was increased by low concentrations of kainate without affecting their amplitudes, suggesting a presynaptic mechanism of action. The facilitatory and inhibitory effects of kainate were mimicked by the GluR5 subunit selective agonist ATPA. In addition to decreasing EPSC amplitudes, high concentrations of kainate and ATPA induced an inward current which was not blocked by AMPA- or NMDA- receptor antagonists GYKI52466 and D-APV, respectively. The inward currents were blocked by the AMPA/KA receptor antagonist CNQX, indicating the presence of postsynaptic kainate receptors. Single shock stimulation in the presence of GYKI52466 and D-APV evoked an EPSC which was blocked by CNQX. The GluR5 antagonist LY382884 changed paired-pulse facilitation to paired pulse depression, indicating that synaptically released glutamate can activate presynaptic kainate receptors. These results suggest that kainate receptors containing GluR5 subunits play a major role in glutamatergic transmission in rat neocortex, having both presynaptic modulatory and direct postsynaptic excitatory actions.

GABAA receptors mediate the majority of inhibitory neurotransmission in the CNS. Genetic deletion of the α1 subunit of GABAA receptors results in a loss of α1-mediated fast inhibitory currents and a marked reduction in density of GABAA receptors. A grossly normal phenotype of α1-deficient mice suggests the presence of neuronal adaptation to these drastic changes at the GABA synapse. We used cDNA microarrays to identify transcriptional fingerprints of cellular plasticity in response to altered GABAergic inhibition in the cerebral cortex and cerebellum of α1 mutants. In silico analysis of 982 mutation-regulated transcripts highlighted genes and functional groups involved in regulation of neuronal excitability and synaptic transmission, suggesting an adaptive response of the brain to an altered inhibitory tone. Public gene expression databases permitted identification of subsets of transcripts enriched in excitatory and inhibitory neurons as well as some glial cells, providing evidence for cellular plasticity in individual cell types. Additional analysis linked some transcriptional changes to cellular phenotypes observed in the knock-out mice and suggested several genes, such as the early growth response 1 (Egr1), small GTP binding protein Rac1 (Rac1), neurogranin (Nrgn), sodium channel β4 subunit (Scn4b), and potassium voltage-gated Kv4.2 channel (Kcnd2) as cell type-specific markers of neuronal plasticity. Furthermore, transcriptional activation of genes enriched in Bergman glia suggests an active role of these astrocytes in synaptic plasticity. Overall, our results suggest that the loss of α1-mediated fast inhibition produces diverse transcriptional responses that act to regulate neuronal excitability of individual neurons and stabilize neuronal networks, which may account for the lack of severe abnormalities in α1 null mutants.

Two functionally distinct forms of synaptic plasticity, Hebbian long-term potentiation (LTP) and homeostatic synaptic scaling, are thought to cooperate to promote information storage and circuit refinement. Both arise through changes in the synaptic accumulation of AMPA receptors (AMPAR), but whether they use similar or distinct receptor trafficking pathways is unknown. Here we show that TTX-induced synaptic scaling in cultured visual cortical neurons leads to the insertion of GluR2-containing AMPARs at synapses. Similarly, visual deprivation with monocular TTX injections results in synaptic accumulation of GluR2-containing AMPARs. Unlike chemical LTP, synaptic scaling is blocked by a GluR2 C-tail peptide, but not by a GluR1 C-tail peptide. Knockdown of endogenous GluR2 with an shRNA also blocks synaptic scaling, but not chemical LTP. Scaling can be rescued with expression of exogenous GluR2 resistant to the shRNA, but a chimeric GluR2 subunit with the C-terminal domain swapped with the GluR1 C-terminal domain (GluR2/CT1) does not rescue synaptic scaling, indicating that regulatory sequences on the GluR2 C-tail are required for the accumulation of synaptic AMPARs during scaling. Taken together, our results suggest that synaptic scaling and LTP utilize different trafficking pathways, making these two forms of plasticity both functionally and molecularly distinct.

The development of a balance between excitatory and inhibitory synapses is a critical process in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity plays an important role in achieving such a balance in the developing cortex, but the mechanism that regulates this process is unknown. During development, GABA, the primary inhibitory neurotransmitter in adults, excites neurons as a result of high expression of the Na+-K+-2Cl− cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in vivo, we show that GABA-induced depolarization is necessary for proper excitatory synapse formation and dendritic development of newborn cortical neurons. Blocking NKCC1 with the diuretic bumetanide during development leads to similar persistent changes in cortical circuitry in the adult. Interestingly, expression of a voltage-independent NMDA receptor rescues the failure of NKCC1 knockdown neurons to develop excitatory AMPA transmission, indicating that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex.

Experience-dependent brain plasticity typically declines after an early critical period during which circuits are established. Loss of plasticity with closure of the critical period limits improvement of function in adulthood, but the mechanisms that change the brain’s plasticity remain poorly understood. Here, we identified an increase in expression of Lynx1 protein in mice that prevented plasticity in the primary visual cortex late in life. Removal of this molecular brake enhanced nicotinic acetylcholine receptor signaling. Lynx1 expression thus maintains stability of mature cortical networks in the presence of cholinergic innervation. The results suggest that modulating the balance between excitatory and inhibitory circuits reactivates visual plasticity and may present a therapeutic target.

Monocular visual deprivation (MD) produces profound changes in the ocular dominance (OD) of neurons in the visual cortex. MD shifts visually evoked responses away from the deprived eye and toward domination by the open-eye. Over 30 years ago, two different theories were proposed to account for these changes: either through effects on excitatory visual drive, thereby shifting the balance of excitation in favor of the open-eye, or through effects on intracortical inhibition, thereby suppressing responses from the deprived eye. In the intervening years, a scientific consensus emerged that the major functional effects of MD result from plasticity at excitatory connections in the visual cortex. A recent study by Yazaki-Sugiyama et al. (2009) in mouse visual cortex appears to re-open the debate. Here we take a critical look at these intriguing new data in the context of other recent findings in rodent visual cortex.

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the AMPA subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor-mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2 and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20±0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26±0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32±0.03. The rectification index (control 2.39±0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02±0.11 and 0.93±0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.

Summary

Light deprivation lowers the threshold for long-term depression (LTD) and long-term potentiation (LTP) in visual cortex by a process termed metaplasticity, but the mechanism is unknown. The decreased LTD/P threshold correlates with a decrease in the ratio of NR2A to NR2B subunits of cortical NMDA receptors (NMDARs) and a slowing of NMDAR-mediated excitatory postsynaptic currents (EPSCs). However, whether and how changes in NR2 subunit expression contribute to LTD and LTP remains controversial. In the present study, we used an NR2A knockout (KO) mouse to examine the role of this subunit in the experience-dependent modulation of NMDAR properties, LTD, and LTP. In wild-type (WT) mice, we found what has previously been shown in rats, that dark rearing alters NMDAR EPSC kinetics and temporal summation, LTP, and LTD in layer 2/3 of visual cortex. Deletion of NR2A mimicked the effect of dark-rearing on NMDAR EPSCs, and there was no additional effect of dark-rearing in the NR2A KO mice. Thus, deletion of NR2A abrogates the effects of visual experience on NMDAR EPSCs. Moreover, metaplasticity of LTP and LTD was completely lost in the absence of NR2A. These data support the hypothesis that experience-dependent changes in NR2A/B are functionally significant and yield a mechanism for an adjustable synaptic modification threshold in visual cortex.

Brain-derived neurotrophic factor (BDNF) regulates synapses, but the distribution of BDNF and its receptor TrkB relative to the location of glutamatergic and γ-aminobutyric acidergic (GABAergic) synapses is presently unknown. Immunocytochemistry was performed in primary hippocampal neuron cultures to determine whether BDNF and TrkB are preferentially localized to excitatory or inhibitory markers at 7, 14, and 21 days in vitro (DIV). Glutamatergic sites were localized with vesicular glutamate transporter type 1 (VGLUT1) as presynaptic marker and the NR1 subunit of the NMDA receptor and the GluR1 subunit of the AMPA receptor as receptor markers. GABAergic sites were labeled with the 65-kDa isoform of glutamic acid decarboxylase (GAD-65) as presynaptic marker and the γ2 subunit of the GABAA receptor as receptor marker. During development, <30% of BDNF punctae and TrkB clusters were localized to glutamatergic and GABAergic markers. Because their rates of colocalization did not change from 7 to 21 DIV, this study details the distribution of BDNF and TrkB at 14 DIV. BDNF was preferentially colocalized with glutamatergic markers VGLUT1 and NR1 (~30% each). TrkB was also relatively highly colocalized with VGLUT1 and NR1 (~20% each) but was additionally highly colocalized with GABAergic markers GAD-65 (~20%) and γ2 (~30%). NR1 clusters colocalized with BDNF puncta and TrkB clusters were mostly extrasynaptic, as were γ2 clusters colocalized with TrkB clusters. These results show that, whereas most BDNF and TrkB protein is extrasynaptic, BDNF is preferentially associated with excitatory markers and that TrkB is associated equally with excitatory and inhibitory markers.

While AMPA-type glutamate receptors (AMPARs) found at principal neuron excitatory synapses typically contain the GluR2 subunit, several forms of behavioral experience have been linked to the de novo synaptic insertion of calcium-permeable (CP) AMPARs defined by their lack of GluR2. In particular, whisker experience drives synaptic potentiation as well as the incorporation of CP-AMPARs in the neocortex. Previous studies implicate PICK1 (protein interacting with C kinase-1) in activity-dependent internalization of GluR2, suggesting one potential mechanism leading to the subsequent accumulation of synaptic CP-AMPARs and increased synaptic strength. Here we test this hypothesis by employing a whisker stimulation paradigm in PICK1 knockout mice. We demonstrate that PICK1 facilitates the surface expression of CP-AMPARs and is indispensible for their experience-dependent synaptic insertion. However, the failure to incorporate CP-AMPARs in PICK1 knockouts does not preclude sensory-induced enhancement of synaptic currents. Our results indicate that synaptic strengthening in the early postnatal cortex does not require PICK1 or the addition of GluR2-lacking AMPARs. Instead, PICK1 permits changes in AMPAR subunit composition to occur in conjunction with synaptic potentiation.

High-resolution diffraction was obtained from GluR4 AMPA-receptor ligand-binding domain crystals using a combination of cryogenic and room-temperature data-collection strategies.

Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (α-­amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryo­protection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 Å resolution. Together, these crystals lay the foundation for a structural comparison of LBD–agonist interactions in distinct AMPA-R subunits.