Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-D-aspartic acid receptor activity

Neurotrophins (NTs) have recently been found to regulate synaptic transmission in the hippocampus. Whole-cell and single-channel recordings from cultured hippocampal neurons revealed a mechanism responsible for enhanced synaptic strength. Specifically, brain-derived neurotrophic factor augmented glutamate-evoked, but not acetylcholine-evoked, currents 3-fold and increased N-methyl-D-aspartic acid (NMDA) receptor open probability. Activation of trkB NT receptors was critical, as glutamate currents were not affected by nerve growth factor or NT-3, and increased open probability was prevented by the tyrosine kinase inhibitor K-252a. In addition, the NMDA receptor antagonist MK-801 blocked brain-derived neurotrophic factor enhancement of synaptic transmission, further suggesting that NTs modulate synaptic efficacy via changes in NMDA receptor function.     

Effect of diffusion potential, osmosis and ion-exchange on transdermal drug delivery: theory and experiments

Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70-80%), NMDA-induced inward currents were inhibited by the adenosine A2A receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the A2A receptor selective antagonist 8-(3-chlorostyryl)caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP-beta-S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14-24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca2+ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, A2A receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca2+ pathway rather than the adenylate cyclase/PKA pathway.     

A postsynaptic interaction between dopamine D1 and NMDA receptors promotes presynaptic inhibition in the rat nucleus accumbens via adenosine release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM)-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 microM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 microM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a long-lasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3 +/- 66.1%) and aspartate (286.3 +/- 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.     

Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase

It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.     

Estradiol-induced enhancement of object memory consolidation involves NMDA receptors and protein kinase A in the dorsal hippocampus of female C57BL/6 mice.

This study examined the role of dorsal hippocampal NMDA receptors and PKA activation in 17β-estradiol (E?)-induced enhancement of object memory consolidation. Mice explored two identical objects during training, after which they immediately received intraperitoneal injections of 0.2 mg/kg E?, and bilateral dorsal hippocampal infusions of Vehicle, the NMDA receptor antagonist APV (2.5 μg/side), or the cAMP inhibitor Rp-cAMPS (18.0 μg/side). Retention was tested 48 hours later. The enhanced object memory and increased ERK phosphorylation observed with E? alone was reduced by APV and Rp-cAMPS, suggesting that estrogenic enhancement of object memory involves NMDA receptors and PKA activation within the dorsal hippocampus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cross-modulation of synaptic plasticity by beta-adrenergic and 5-HT1A receptors in the rat basolateral amygdala

Neurotransmitter receptors are often colocalized in a neuron with other receptors, and activation of one receptor can either amplify or antagonize the response to a colocalized receptor. The aim of this study was to investigate the cross-regulation of synaptic transmission by beta-adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of presynaptic beta-adrenergic receptors with isoproterenol (Iso) in the basolateral amygdala resulted in a long-lasting increase in synaptic transmission. This effect was mimicked by forskolin, an activator for adenylyl cyclase and a cAMP analog. In addition, the effect of forskolin was blocked by catalytic and regulatory site antagonists for cAMP-dependent protein kinase (PKA), indicating a PKA-mediated mechanism. Application of 5-HT depressed the synaptic transmission and blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To determine the locus of interaction, Sp-cAMPS, a membrane-permeable activator of PKA, was applied, and the potentiation produced by Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These results suggest that the interaction between the intracellular signaling pathways activated by 5-HT1A and beta-adrenergic receptors occurs at a step downstream from cAMP production.     

Adrenergic blockade in diabetic and uninephrectomized rats: effects on renal size and on renal and urinary contents of epidermal growth factor

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D-aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract.     

Neuromodulation of dendritic action potentials

The extent to which regenerative action potentials invade hippocampal CA1 pyramidal dendrites is dependent on both recent activity and distance from the soma. Previously, we have shown that the amplitude of back-propagating dendritic action potentials can be increased by activating either protein kinase A (PKA) or protein kinase C (PKC) and a subsequent depolarizing shift in the activation curve for dendritic K+ channels. Physiologically, an increase in intracellular PKA and PKC would be expected upon activation of beta-adrenergic and muscarinic acetylcholine receptors, respectively. Accordingly, we report here that activation of either of these neurotransmitter systems results in an increase in dendritic action-potential amplitude. Activation of the dopaminergic neurotransmitter system, which is also expected to raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and PKA levels, increased action-potential amplitude in only a subpopulation of neurons tested.     

Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits. Yet, the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.     

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons

Astrocytes exhibit a form of excitability and communication on the basis of intracellular Ca2+ variations (Cornell-Bell et al., 1990; Charles et al., 1991) that can be initiated by neuronal activity (Dani et al., 1992; Porter and McCarthy, 1996). A Ca2+ elevation in astrocytes induces the release of glutamate (Parpura et al., 1994; Pasti et al., 1997; Araque et al., 1998;Bezzi et al., 1998), which evokes a slow inward current in neurons and modulates action potential-evoked synaptic transmission between cultured hippocampal cells (Araque et al., 1998), suggesting that astrocytes and neurons may function as a network with bidirectional communication. Here we show that a Ca2+ elevation in astrocytes increases the frequency of excitatory as well as inhibitory miniature postsynaptic currents (mPSCs), without modifying their amplitudes. Thapsigargin incubation, microinjection of the Ca2+ chelator BAPTA, and photolysis of the Ca2+ cage NP-EGTA demonstrate that a Ca2+ elevation in astrocytes is both necessary and sufficient to modulate spontaneous transmitter release. This Ca2+-dependent release of glutamate from astrocytes enhances mPSC frequency by acting on NMDA glutamate receptors, because it is antagonized by D-2-amino-5-phosphonopentanoic acid (AP5) or extracellular Mg2+. These NMDA receptors are located extrasynaptically, because blockage specifically of synaptic NMDA receptors by synaptic activation in the presence of the open channel blocker MK-801 did not impair the AP5-sensitive astrocyte-induced increase of mPSC frequency. Therefore, astrocytes modulate spontaneous excitatory and inhibitory synaptic transmission by increasing the probability of transmitter release via the activation of NMDA receptors.     

Enhancement of NMDA receptor-mediated synaptic potential by isoproterenol is blocked by Rp-adenosine 3'',5''-cyclic monophosphothioate

The intracellular mechanisms underlying the facilitatory action of isoproterenol (Iso) on the NMDA receptor-mediated synaptic potential (EPSPNMDA) was investigated in an in vitro slice preparation of rat amygdala. Intracellular recordings were made from basolateral amygdala neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and picrotoxin (50 microM) which block non-NMDA and GABAA receptors, respectively. Superfusion of Iso (15 microM) produced a sustained increase in EPSPNMDA. Rp-adenosine-3',5'-cyclic monophosphotioate (Rp-cAMPS), a potent inhibitor of protein kinase A (PKA) alone decreased the amplitude of EPSPNMDA below baseline values and prevented the subsequent potentiation by Iso. Forskolin, a direct activator of adenylate cyclase, mimics the effect of Iso, and Rp-cAMPS also reversed forskolin-induced enhancement of EPSNMDA. These results suggest that cAMP-dependent protein kinase mediates the enhancement of EPSPNMDA by Iso in the amygdala.     

Estimating the contributions of NMDA and non-NMDA currents to EPSPs in retinal ganglion cells

Whole-cell recordings were obtained from retinal ganglion cells of the tiger salamander (Ambystoma tigrinum) in a superfused slice preparation to evaluate contributions of NMDA (N-methyl-D-aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid) receptors to excitatory postsynaptic potentials (EPSPs) of retinal ganglion cells. Synaptic activation of retinal ganglion cells was achieved through the use of a brief pressure pulse of hyperosmotic Ringer (Ringer + sucrose) delivered through a microelectrode visually placed in the inner plexiform layer while whole-cell recordings were obtained from adjacent cells in the ganglion cell layer. Separation of NMDA and KA/AMPA excitatory postsynaptic currents (EPSCs) was achieved through the application of the antagonists NBQX and D-AP7, while inhibitory currents were blocked by strychnine and picrotoxin. Simple addition of the two independent EPSCs showed, most often, that the sum of the KA/AMPA and NMDA currents was less than the control response, but in some cases the sum of the two currents exceeded the magnitude of the control response. Neither result was consistent with expectations based on voltage-clamp principles and the assumption that the two currents were independent; for this reason, we considered the possibility of nonlinear interactions between KA/AMPA and NMDA receptors. Computer simulations were carried out to evaluate the summation experiments. We used both an equivalent cylinder model and a more realistic, compartmental model of a ganglion cell constrained by a passive leakage conductance, a linear KA/AMPA synaptic current, and a nonlinear NMDA current based on the well-known, voltage-sensitive Mg2+ block. Computer simulation studies suggest that the hypo- and hyper-summation of NMDA and KA/AMPA currents, observed physiologically, can be accounted for by a failure to adequately space clamp the neuron. Clamp failure leads to enhanced NMDA currents as the ion channels are relieved of the Mg2+ block; their contribution is thus exaggerated depending on the magnitude of the conductance change and the spatial location of the synaptic input.     

NMDA receptor properties in rat supraoptic magnocellular neurons: characterization and postnatal development

Hypothalamo-neurohypophysial magnocellular neurons display specific electrical activities in relation to the mode of release of their hormonal content (vasopressin or oxytocin). These activities are under strong glutamatergic excitatory control. The implication of NMDA receptors in the control of vasopressinergic and oxytocinergic neurons is still a matter of debate. We here report the first detailed characterization of functional properties of NMDA receptors in voltage-clamped magnocellular neurons acutely dissociated from the supraoptic nucleus. All cells responded to NMDA with currents that reversed polarity around 0 mV and were inhibited by D-2-amino-5-phosphonovalerate (D-APV) and by 100 microM extracellular Mg2+ (at -80 mV). Sensitivity to the co-agonist glycine (EC50, 2 microM) was low compared with most other neuronal preparations. The receptors displayed low sensitivity to ifenprodil, were insensitive to glycine-independent potentiation by spermine, and had a unitary conductance of 50 pS. No evidence was found for two distinct cell populations, suggesting that oxytocinergic and vasopressinergic neurons express similar NMDA receptors. Characterization of NMDA receptors at different postnatal ages revealed a transient increase in density of NMDA currents during the second postnatal week. This was accompanied by a specific decrease in sensitivity to D-APV, with no change in NMDA sensitivity or any other properties studied. Supraoptic NMDA receptors thus present characteristics that strikingly resemble those of reconstituted receptors composed of NR1 and NR2A subunits. Understanding the functional significance of the development of NMDA receptors in the supraoptic nucleus will require further knowledge about the maturation of neuronal excitability, synaptic connections and neurohormone release mechanisms.     

Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K+ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (PCa/PNa = 5.6 +/- 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, PCa/PNa, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 microM, 240 nM, and 81 microM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed wide-spread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit.     

The role of dorsal hippocampus and basolateral amygdala NMDA receptors in the acquisition and retrieval of context and contextual fear memories.

The authors used 3-phase context preexposure facilitation methodology to study the contribution of N-methyl-D-aspartate (NMDA) receptors in dorsal hippocampus (DH) and the basal lateral region of the amygdala (BLA) to (a) acquisition of the context memory, (b) retrieval of the context memory, (c) acquisition of context-shock association, and (d) retrieval of the context-shock association. The NMDA receptor antagonist D-2-amino-5 phosphonopentanoic acid (D-AP5) was injected into either the DH or BLA prior to (a) the context preexposure phase, (b) the immediate shock phase, or (c) the test for contextual fear. Antagonizing NMDA receptors in the DH impaired the acquisition of the context memory but did not affect its retrieval or retrieval of the fear memory. Antagonizing NMDA receptors with D-AP5 in the BLA impaired acquisition of the context-shock association but had no effect on the expression of fear. However, both DL-AP5 and L-AP5 reduced the expression of fear when they were injected into the amygdala prior to testing for contextual fear. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.     

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala. Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium. Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABA(A)-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning.     

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.