Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.     

Regulation of AMPA receptor lateral movements

An essential feature in the modulation of the efficacy of synaptic transmission is rapid changes in the number of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors at post-synaptic sites on neurons. Regulation of receptor endo- and exocytosis has been shown to be involved in this process. Whether regulated lateral diffusion of receptors in the plasma membrane also participates in receptor exchange to and from post-synaptic sites remains unknown. We analysed the lateral mobility of native AMPA receptors containing the glutamate receptor subunit GluR2 in rat cultured hippocampal neurons, using single-particle tracking and video microscopy. Here we show that AMPA receptors alternate within seconds between rapid diffusive and stationary behaviour. During maturation of neurons, stationary periods increase in frequency and length, often in spatial correlation with synaptic sites. Raising intracellular calcium, a central element in synaptic plasticity, triggers rapid receptor immobilization and local accumulation on the neuronal surface. We suggest that calcium influx prevents AMPA receptors from diffusing, and that lateral receptor diffusion to and from synaptic sites acts in the rapid and controlled regulation of receptor numbers at synapses.     

Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus.

Neurotransmission at most excitatory synapses in the brain operates through two types of glutamate receptor termed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors; these mediate the fast and slow components of excitatory postsynaptic potentials respectively. Activation of NMDA receptors can also lead to a long-lasting modification in synaptic efficiency at glutamatergic synapses; this is exemplified in the CA1 region of the hippocampus, where NMDA receptors mediate the induction of long-term potentiation (LTP). It is believed that in this region LTP is maintained by a specific increase in the AMPA receptor-mediated component of synaptic transmission. We now report, however, that a pharmacologically isolated NMDA receptor-mediated synaptic response can undergo robust, synapse-specific LTP. This finding has implications for neuropathologies such as epilepsy and neurodegeneration, in which excessive NMDA receptor activation has been implicated. It adds fundamentally to theories of synaptic plasticity because NMDA receptor activation may, in addition to causing increased synaptic efficiency, directly alter the plasticity of synapses.     

Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2

Synaptic transmission from excitatory nerve cells in the mammalian brain is largely mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors located at the surface of dendritic spines. The abundance of postsynaptic AMPA receptors correlates with the size of the synapse and the dimensions of the dendritic spine head. Moreover, long-term potentiation is associated with the formation of dendritic spines as well as synaptic delivery of AMPA receptors. The molecular mechanisms that coordinate AMPA receptor delivery and spine morphogenesis are unknown. Here we show that overexpression of the glutamate receptor 2 (GluR2) subunit of AMPA receptors increases spine size and density in hippocampal neurons, and more remarkably, induces spine formation in GABA-releasing interneurons that normally lack spines. The extracellular N-terminal domain (NTD) of GluR2 is responsible for this effect, and heterologous fusion proteins of the NTD of GluR2 inhibit spine morphogenesis. We propose that the NTD of GluR2 functions at the cell surface as part of a receptor-ligand interaction that is important for spine growth and/or stability.     

Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.     

Long-term potentiation of electrotonic coupling at mixed synapses

Long-term potentiation of chemical synapses is closely related to memory and learning. Studies of this process have concentrated on chemically mediated excitatory synapses. By contrast, activity-dependent modification of gap junctions, which also widely exist in higher structures such as hippocampus and neocortex, has not been described. Here we report that at mixed synapses between sensory afferents and an identified reticulospinal neuron, the electrotonic coupling potential can be potentiated, as well as the chemically mediated excitatory postsynaptic potential, for a prolonged time period using a stimulation paradigm like that which produces long-term potentiation in hippocampus. The effect on coupling is due to an increase in gap-junctional conductance. Our data indicate that the potentiation of both synaptic components requires an increase in intracellular calcium, involves activation of NMDA (N-methyl-D-aspartate) receptors, and is specific to the tetanized pathway.     

NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats

Acidic amino acids, such as glutamate and aspartate, are thought to be excitatory transmitters in the cerebral neocortex and hippocampus. Receptors for these amino acids can be classified into at least three types on the basis of their agonists. Quisqualate-preferring receptors and kainate-preferring receptors are implicated in the mediation of synaptic transmission in many regions including the hippocampus and visual cortex, whereas N-methyl-D-aspartate (NMDA)-preferring receptors are thought to be involved in modulating synaptic efficacy, for example in longterm potentiation, a form of synaptic plasticity in the hippocampus. In the visual cortex of the cat and monkey, it is well established that synaptic plasticity, estimated by susceptibility of binocular responsiveness of cortical neurons to monocular visual deprivation, disappears after the 'critical' period of postnatal development. Here we report that during the critical period in young kittens, a selective NMDA-receptor antagonist blocks visual responses of cortical neurons much more effectively than it does in the adult cat. This suggests that NMDA receptors may be involved in establishing synaptic plasticity in the kitten visual cortex.     

Cloning of a putative high-affinity kainate receptor expressed predominantly in hippocampal CA3 cells.

Kainic acid is a potent neurotoxin for certain neurons. Its neurotoxicity is thought to be mediated by an excitatory amino-acid-gated ion channel (ionotropic receptor) possessing nanomolar affinity for kainate. Here we describe a new member of the rat excitatory amino-acid receptor gene family, KA-1, that has a 30% sequence similarity with the previously characterized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR-A to -D. The pharmacological profile of expressed recombinant KA-1 determined in binding experiments with [3H]kainate is different from that of the cloned AMPA receptors and similar to the mammalian high-affinity kainate receptor (kainate greater than quisqualate greater than glutamate much greater than AMPA) with a dissociation constant of about 5 nM for kainate. The selectively high expression of KA-1 messenger RNA in the CA3 region of the hippocampus closely corresponds to autoradiographically located high-affinity kainate binding sites. This correlation, as well as the particular in vivo pattern of neurodegeneration observed on kainate-induced neurotoxicity, suggests that KA-1 participates in receptors mediating the kainate sensitivity of neurons in the central nervous system.     

Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses.

Most of the fast excitatory synaptic transmission in the mammalian brain is mediated by ionotrophic glutamate receptors, of which there are three subtypes: AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate), NMDA (N-methyl-D-aspartate) and kainate. Although kainate-receptor subunits (GluR5-7, KA1 and 2) are widely expressed in the mammalian central nervous system, little is known about their function. The development of pharmacological agents that distinguish between AMPA and kainate receptors has now allowed the functions of kainate receptors to be investigated. The modulation of synaptic transmission by kainate receptors and their synaptic activation in a variety of brain regions have been reported. The expression of kainate receptor subunits is developmentally regulated but their role in plasticity and development is unknown. Here we show that developing thalamocortical synapses express postsynaptic kainate receptors as well as AMPA receptors; however, the two receptor subtypes do not colocalize. During the critical period for experience-dependent plasticity, the kainate-receptor contribution to transmission decreases; a similar decrease occurs when long-term potentiation is induced in vitro. This indicates that during development there is activity-dependent regulation of the expression of kainate receptors at thalamocortical synapses.     

Synaptic localization of kainic acid binding sites

The heterocyclic compound kainic acid (KA) is a potent excitant when applied to mammalian neurones. Lesions caused by injections of KA into the rat striatum and hippocampus cause similar patterns of damage to those seen in Huntington's chorea and status epilepticus, respectively. Although it was originally thought to be a glutamate agonist, it is now clear that KA does not act on the majority of the receptors for glutamate, and in fact seems to act on a class of receptors which are distinct from those which mediate responses to other excitatory amino acids. The potent and selective neurotoxic effects of this compound may be mediated by these same receptors. At present, the relative distribution of junctional and extrajunctional (non-synaptic) receptors is unknown and resolution of this issue would provide important insights into the action of KA on the central nervous system (CNS). We show here that KA binding sites are greatly enriched in isolated synaptic junctions from rat brain and, using an in vitro autoradiographic technique, we have found that these binding sites are concentrated specifically in terminal fields where KA acts as a potent neurotoxin.     

ATP mediates fast synaptic transmission in mammalian neurons.

In addition to its diverse functions inside cells, ATP can act at several types of cell-surface receptor. One of these (P2X-purinoceptor) is believed to be a ligand-gated cation channel. The presence of P2X receptors on autonomic, sensory and central neurons suggests that ATP might be released to act as a fast excitatory synaptic transmitter. Here we record excitatory synaptic potentials and currents from cultured coeliac ganglion neurons which are mimicked by ATP, blocked by the P2-purinoceptor antagonist suramin, desensitized by alpha,beta-methylene-ATP and unaffected by antagonists acting at nicotine, 5-hydroxytryptamine, N-methyl-D-aspartate (NMDA), non-NMDA glutamate, gamma-aminobutyric acid (GABA), noradrenaline or adenosine receptors. We conclude that ATP is the neurotransmitter at this neuroneuronal synapse.     

ATP receptor-mediated synaptic currents in the central nervous system.

Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors.     

Anatomical distributions of four pharmacologically distinct 3H-L-glutamate binding sites

Glutamate is thought to serve as a major excitatory neurotransmitter throughout the central nervous system (CNS); electrophysiological studies indicate that its action is mediated by multiple receptors. Four receptors have been characterized by their selective sensitivity to N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA) and 2-amino-4-phosphonobutyric acid (APB). Electrophysiological evidence indicates that these receptors are all present in the rat hippocampus and that the anatomically discrete synaptic fields within the hippocampus exhibit differential sensitivity to the selective excitatory amino acid agents. Thus, we have used the hippocampus as a model system to investigate possible subpopulations of 3H-L-glutamate binding sites. By using quantitative autoradiography, the pharmacological specificity of 3H-L-glutamate binding in discrete terminal fields was determined. We report here that there are at least four distinct classes of 3H-L-glutamate binding sites which differ in their anatomical distribution, pharmacological profile and regulation by ions. Two of these sites seem to correspond to the KA and NMDA receptor classes, and a third site may represent the QA receptor. The fourth binding site does not conform to present receptor classifications. None of these binding sites corresponds to the major glutamate binding site observed in biochemical studies.     

Multiple classes of glutamate receptor on depolarizing bipolar cells in retina

Multiple subtypes of excitatory amino acid receptor have been found on individual dissociated neurones. These findings were obtained from cells without intact synaptic connections, so the functional roles for such receptor subtypes are unknown. We have recorded intracellular responses from depolarizing bipolar cells (DBC) that receive direct synaptic input from two distinct populations of neurones: rods and cones. We report here that 2-amino-4-phosphonobutyrate (APB), a glutamate analogue, reveals two subtypes of glutamate receptors on DBCs. APB acts on the same receptor that mediates synaptic transmission from rods but has no action on the second subtype of glutamate receptor. These results show that the rod and cone inputs to DBCs are mediated by pharmacologically distinct receptors and that subtypes of glutamate receptor existing on single neurones can subserve separate, functionally defined synaptic inputs.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving

Relapse to cocaine use after prolonged abstinence is an important clinical problem. This relapse is often induced by exposure to cues associated with cocaine use. To account for the persistent propensity for relapse, it has been suggested that cue-induced cocaine craving increases over the first several weeks of abstinence and remains high for extended periods. We and others identified an analogous phenomenon in rats that was termed 'incubation of cocaine craving': time-dependent increases in cue-induced cocaine-seeking over the first months after withdrawal from self-administered cocaine. Cocaine-seeking requires the activation of glutamate projections that excite receptors for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the nucleus accumbens. Here we show that the number of synaptic AMPA receptors in the accumbens is increased after prolonged withdrawal from cocaine self-administration by the addition of new AMPA receptors lacking glutamate receptor 2 (GluR2). Furthermore, we show that these new receptors mediate the incubation of cocaine craving. Our results indicate that GluR2-lacking AMPA receptors could be a new target for drug development for the treatment of cocaine addiction. We propose that after prolonged withdrawal from cocaine, increased numbers of synaptic AMPA receptors combined with the higher conductance of GluR2-lacking AMPA receptors causes increased reactivity of accumbens neurons to cocaine-related cues, leading to an intensification of drug craving and relapse.     

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.     

Kainate receptors are involved in synaptic plasticity

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission.     

Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular transporters. Here we show that expression of BNPI, a vesicle-bound transporter associated with sodium-dependent phosphate transport, results in glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by glutamate as it is blocked by a combination of the glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular glutamate transporter and that expression of BNPI suffices to define a glutamatergic phenotype in neurons.     

Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.