Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-d-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using in vivo microdialysis we measured conversion of l-[14C]arginine to l-[14C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome c) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.

A new strategy to decrease N-methyl-d-aspartate (NMDA) receptor coactivation: Inhibition of d-serine synthesis by converting serine racemase into an eliminase

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Dehydroepiandrosterone: A potential signalling molecule for neocortical organization during development

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) are the most abundant steroids produced by the human adrenal, but no receptors have been identified for these steroids, and no function for them has been established, other than as precursors for sex steroid synthesis. DHEA and DHEAS are found in brains from many species, and we have shown that enzymes crucial for their synthesis, especially P450c17 (17α-hydroxylase/c17,20 lyase), are expressed in a developmentally regulated, region-specific fashion in the developing rodent brain. One region of embryonic expression of P450c17, the neocortical subplate, has been postulated to play a role in guiding cortical projections to their appropriate targets. We therefore determined if products of P450c17 activity, DHEA and DHEAS, regulated the motility and/or growth of neocortical neurons. In primary cultures of mouse embryonic neocortical neurons, DHEA increased the length of neurites containing the axonal marker Tau-1, and the incidence of varicosities and basket-like process formations in a dose-dependent fashion. These effects could be seen at concentrations normally found in the brain. By contrast, DHEAS had no effect on Tau-1 axonal neurites but increased the length of neurites containing the dendritic marker microtubule-associated protein-2. DHEA rapidly increased free intracellular calcium via activation of N-methyl-d-aspartate (NMDA) receptors. These studies provide evidence of mechanisms by which DHEA and DHEAS exert biological actions, show that they have specific functions other than as sex steroid precursors, mediate their effects via non-classic steroid hormone receptors, and suggest that their developmentally regulated synthesis in vivo may play crucial and different roles in organizing the neocortex.

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

A neuromodulatory role of interleukin-1β in the hippocampus

It is widely accepted that interleukin-1β (IL-1β), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we show in vivo and in vitro that IL-1β gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with the N-methyl-d-aspartate (NMDA) receptor antagonist, (±)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1β in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.

Carboxyfullerenes as neuroprotective agents

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 μM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Aβ1–42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing dl-aminophosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, β-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of β-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

Response-reinforcement learning is dependent on N-methyl-d-aspartate receptor activation in the nucleus accumbens core

The nucleus accumbens, a site within the ventral striatum, is best known for its prominent role in mediating the reinforcing effects of drugs of abuse such as cocaine, alcohol, and nicotine. Indeed, it is generally believed that this structure subserves motivated behaviors, such as feeding, drinking, sexual behavior, and exploratory locomotion, which are elicited by natural rewards or incentive stimuli. A basic rule of positive reinforcement is that motor responses will increase in magnitude and vigor if followed by a rewarding event. It is likely, therefore, that the nucleus accumbens may serve as a substrate for reinforcement learning. However, there is surprisingly little information concerning the neural mechanisms by which appetitive responses are learned. In the present study, we report that treatment of the nucleus accumbens core with the selective competitive N-methyl-d-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5; 5 nmol/0.5 μl bilaterally) impairs response-reinforcement learning in the acquisition of a simple lever-press task to obtain food. Once the rats learned the task, AP-5 had no effect, demonstrating the requirement of NMDA receptor-dependent plasticity in the early stages of learning. Infusion of AP-5 into the accumbens shell produced a much smaller impairment of learning. Additional experiments showed that AP-5 core-treated rats had normal feeding and locomotor responses and were capable of acquiring stimulus-reward associations. We hypothesize that stimulation of NMDA receptors within the accumbens core is a key process through which motor responses become established in response to reinforcing stimuli. Further, this mechanism, may also play a critical role in the motivational and addictive properties of drugs of abuse.

Dynamin-dependent endocytosis of ionotropic glutamate receptors

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.