Differential regulation of STP, LTP and LTD by structurally diverse NMDA receptor subunit-specific positive allosteric modulators.

Different types of memory are thought to rely on different types of synaptic plasticity, many of which depend on the activation of the N-Methyl-D Aspartate (NMDA) subtype of glutamate receptors. Accordingly, there is considerable interest in the possibility of using positive allosteric modulators (PAMs) of NMDA receptors (NMDARs) as cognitive enhancers. Here we firstly review the evidence that NMDA receptor-dependent forms of synaptic plasticity: short-term potentiation (STP), long-term potentiation (LTP) and long-term depression (LTD) can be pharmacologically differentiated by using NMDAR ligands. These observations suggest that PAMs of NMDAR function, depending on their subtype selectivity, might differentially regulate STP, LTP and LTD. To test this hypothesis, we secondly performed experiments in rodent hippocampal slices with UBP714 (a GluN2A/2B preferring PAM), CIQ (a GluN2C/D selective PAM) and UBP709 (a pan-PAM that potentiates all GluN2 subunits). We report here, for the first time, that: (i) UBP714 potentiates sub-maximal LTP and reduces LTD; (ii) CIQ potentiates STP without affecting LTP; (iii) UBP709 enhances LTD and decreases LTP. We conclude that PAMs can differentially regulate distinct forms of NMDAR-dependent synaptic plasticity due to their subtype selectivity.

Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones.

The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.

Plasticity of hippocampal circuitry in Alzheimer's disease.

Two markers of neuronal plasticity were used to compare the response of the human central nervous system to neuronal loss resulting from Alzheimer's disease with the response of rats to a similar neuronal loss induced by lesions. In rats that had received lesions of the entorhinal cortex, axon sprouting of commissural and associational fibers into the denervated molecular layer of the dentate gyrus was paralleled by a spread in the distribution of tritiated kainic acid-binding sites. A similar expansion of kainic acid receptor distribution was observed in hippocampal samples obtained postmortem from patients with Alzheimer's disease. An enhancement of acetylcholinesterase activity in the dentate gyrus molecular layer, indicative of septal afferent sprouting, was also observed in those patients with a minimal loss of cholinergic neurons. These results are evidence that the central nervous system is capable of a plastic response in Alzheimer's disease. Adaptive growth responses occur along with the degenerative events.

NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones.

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC(50) of 0.36 microm and a maximum inhibition of 73.5 +/- 2.7% (10 microm), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC(50) of 6.2 microm. During postnatal development, the maximum inhibitory effect of 10 microm ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn(2+) (200 nM) or potentiated by the Zn(2+) chelator TPEN (1 microm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.

N-methyl-D-aspartate receptors and Alzheimer's disease.

The results of several studies now suggest that the density of N-methyl-D-aspartate (NMDA) receptors is maintained in many Alzheimer's disease (AD) cases, although loss of these receptors can occur in specific regions as a consequence of severe neuronal loss. Recent findings demonstrate that there are at least two subtypes of the NMDA receptor which are allosterically regulated. To determine the status of the NMDA receptor in AD, studies are required which will examine the activation state of the NMDA receptor and the properties of subtypes in relation to neuronal density and structure.

Plastic response of hippocampal excitatory amino acid receptors to deafferentation and reinnervation.

In vitro autoradiography was used to examine the response of excitatory amino acid receptors in the hippocampus of rat following unilateral lesions of the entorhinal cortex. The density of N-methyl-D-aspartate and quisqualate receptor binding was determined on days 1, 3, 7, 14, 21, 30 and 60 postlesion both ipsilateral and contralateral to the lesion and in unoperated controls. The results are compared to the time-course of deafferentation and reinnervation. The molecular layer of the dentate gyrus contralateral to the lesion is only minimally denervated, but is known to exhibit extensive synapse loss and replacement. N-Methyl-D-aspartate receptor binding density in the contralateral hippocampus increased (10-15% relative to unoperated controls) as early as 3 days postlesion and remained elevated through all postlesion times examined. In contrast, the quisqualate receptors in the contralateral hippocampus were unaffected at all times investigated. In the deafferented molecular layer of the ipsilateral dentate gyrus there was a small transient decrease (15-20%) in the binding density of quisqualate receptors 3 days postlesion. At later postoperative times (30-60 days postlesion) the density of both N-methyl-D-aspartate and quisqualate receptors in the ipsilateral molecular layer was higher (15-50%) than that of unoperated controls. These results indicate that N-methyl-D-aspartate and quisqualate receptors are differentially regulated in response to deafferentation. The rapid decrease in quisqualate (and perhaps also N-methyl-D-aspartate) receptor binding at 3 days postlesion may simply reflect the loss of presynaptic receptors, the turnover of postsynaptic receptors or the down-regulation of postsynaptic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival and functional demonstration of interregional pathways in fore/midbrain slice explant cultures.

An important general question in neurobiology concerns the development and expression of the rich context of neuronal phenotypes, especially in relation to the diverse patterns of connectivity. Organotypic cultures of brain slices may offer distinct advantages for such studies if such a preparation survives, maintains a wide diversity of neuronal phenotypes and displays appropriate synaptic connections between regions. To address these requirements, we utilized long-term organotypic cultures of intact horizontal slices of rat forebrain and midbrain and assessed a variety of markers of phenotype in combination with functional tests of connectivity. This explant preparation displayed a distinct viability requirement such that the greatest explant survival was seen in slices taken from pups of less than postnatal day 7 and was independent of N-methyl-D-aspartate channel blockade. The anatomical features of the major brain regions (e.g., neocortex, striatum, septum, hippocampus, diencephalon and midbrain) were observed in their normal boundaries. The presence of cholinergic and catecholaminergic neurons was demonstrated with acetylcholinesterase histochemistry and tyrosine hydroxylase immunohistochemistry. Labelled neurons displayed multiple, regionally-appropriate cytoarchitectures and, in some cases, could be seen to project to brain regions in a manner quite similar to that seen in vivo. Finally, the direct demonstration of spontaneous and evoked interregional excitatory synaptic transmission was made using whole-cell patch-clamp recordings from striatal neurons which revealed an intact glutamate-using corticostriatal pathway. This simple explant preparation appears to contain a rich diversity of neuronal types and synaptic organization. Therefore, this preparation appears to have several distinct advantages for basic neurobiologic research since it combines long-term culture viability and many features of mature brain including complex interregional neuronal systems.

Autoradiographic characterization of putative excitatory amino acid transport sites.

Removal of excitatory amino acids from the extracellular space is now postulated to occur through at least two distinct transport systems that are distinguished by their ionic dependency. Thus, both sodium-dependent and chloride-dependent systems have been described in the mammalian central nervous system. In this report we attempt to characterize these sites by autoradiography, using D-[3H]aspartate and L-[3H]glutamate as ligands. Previous studies have shown that sequestration of radioligand into membrane vesicles can be a potential artifact when examining transport sites. We have found that sequestration can be alleviated by incubation of tissue sections in xylenes prior to incubation with radioligand. Using in vitro autoradiography we have characterized the two binding sites with respect to their distribution, kinetics and pharmacology. Both appeared to have a single, saturable binding site with Kds in the low micromolar range. Sodium-dependent D-aspartate binding predominated, having a Bmax that was five times greater than chloride-dependent L-glutamate binding in whole brain. The levels of binding to the two sites varied between brain regions. Sodium-dependent D-aspartate binding was highest in the cerebellar molecular layer greater than dentate gyrus molecular layer greater than entorhinal cortex. Chloride-dependent L-glutamate binding was highest in the outer layers of cerebral cortex greater than dentate gyrus molecular layer greater than entorhinal cortex greater than striatum. Pharmacological characterization of these sites also showed major differences. Sodium-dependent D-aspartate binding was most potently inhibited by L-aspartate greater than threo-beta-hydroxyaspartate greater than L-cysteine sulfinic acid greater than L-cysteic acid. Chloride-dependent glutamate binding was most potently inhibited by L-glutamate greater than L-alpha-amino adipic acid greater than quisqualate greater than L-serine-o-sulfate. The differences in distribution, ligand binding properties and pharmacology of these sites suggest that a significant variable in excitatory amino acid circuitry may include heterogeneity in transporters associated with excitatory pathways.

The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography.

The distribution of [3H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.

Glutamate receptors and phosphoinositide metabolism: stimulation via quisqualate receptors is inhibited by N-methyl-D-aspartate receptor activation.

Excitatory amino acid receptors in the neonatal rat hippocampus have opposing actions on phosphoinositide (PI) metabolism. Quisqualic acid (QA), but not the QA receptor agonist AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), potently stimulates inositol phosphate (IP) formation. Activation of NMDA (N-methyl-D-aspartate) receptors inhibits the QA-induced stimulation by 70% by a mechanism which is dependent on extracellular calcium.

L-[3H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis.

The anatomical distribution of L-[3H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-[3H]glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of Ca2+, Cl- and Na+ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-[3H]2-amino-5-phosphonopentanoate (D-[3H]AP5), [3H]kainate ([3H]KA) and [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.

Distribution of [3H]AMPA binding sites in rat brain as determined by quantitative autoradiography.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [3H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I-III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [3H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.

The effect of entorhinal cortical ablation on the distribution of muscarinic cholinergic receptors in the rat hippocampus.

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.

Cations differentially affect subpopulations of L-glutamate receptors in rat synaptic plasma membranes.

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.

A comparison of 2-amino-4-phosphonobutyric acid (AP4) receptors and [3H]AP4 binding sites in the rat brain.

The glutamate analogue 2-amino-4-phosphonobutyric acid (AP4) is a potent antagonist at several synapses where an excitatory amino acid appears to be the neurotransmitter. Previous studies identified a Cl-/Ca2+ dependent [3H]glutamate binding site in synaptic plasma membrane (SPM) preparations that was also labeled by [3H]AP4 and exhibited a pharmacology similar to the AP4 receptor. This report examines the pharmacological specificity in both biochemical and electrophysiological preparations in greater detail. Several compounds are identified which readily interact with the apparent binding site in membranes, but neither mimic nor inhibit the action of AP4 in electrophysiological studies. The rate of dissociation of [3H]AP4 from SPMs is shown to increase in the presence of added AP4 and increasing the osmolarity in the SPM binding assay decreases the level of observed [3H]AP4 binding. These findings indicate both a heterogeneous population of binding sites and the occurrence of transport. It is concluded that much of the AP4 binding observed in SPM preparations is to a site other than the AP4 receptor. The results provide a further pharmacological description of AP4 receptors which should facilitate the identification of the receptor in biochemical preparations.

Action of 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP): a new and highly potent antagonist of N-methyl-D-aspartate receptors in the hippocampus.

A new compound, 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces L-[3H]glutamate binding in regions of the hippocampus rich in N-methyl-D-aspartate (NMDA) receptors, but not in regions rich in kainate sites. In isolated membrane fraction preparations, CPP displaces L-[3H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA greater than homocysteate greater than aspartate greater than glutamate greater than kainate greater than or equal to quisqualate. CPP has no effect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synaptic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compound is, therefore, a highly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.

Regional distribution and ionic requirement of Cl-/Ca2+-activated and Cl-/Ca2+-independent glutamate receptors in rat brain.

Recent studies have shown that Cl- and Ca2+ ions increase [3H]glutamate binding to rat forebrain synaptic plasma membranes by expressing a new class of glutamate receptors. We examined the regional distribution of these two classes of glutamate binding sites and further characterized their ionic requirements. Significant differences in both Cl-/Ca2+-independent (basal) and Cl-/Ca2+-activated receptors, as well as the ratios of these two receptor classes were observed among different areas of the CNS. Cl- and Ca2+ appeared to act synergistically, with Cl-ion an absolute requirement for Ca2+ stimulation, in expressing these additional binding sites. Ca2+ alone did not affect glutamate binding.

Synthesis of [3H]2-amino-4-phosphonobutyric acid and characterization of its binding to rat brain membranes: a selective ligand for the chloride/calcium-dependent class of L-glutamate binding sites.

[3H]2-amino-4-phosphonobutyric acid was synthesized by the conjugate addition of 1-lithio-2-trimethylsilyethyne to diethyl ethynylphosphate followed by catalytic tritiation and hydrolysis. Radiolabelled 2-amino-4-phosphonobutyric acid binds to a distinct class of L-glutamate binding sites and does not exhibit appreciable binding to sites not displaced by L-glutamate. The binding affinity (Kd = 5.1 +/- 0.4 microM) and pharmacological profile correspond to those values obtained from physiological studies of 2-amino-4-phosphonobutyric acid inhibition of synaptic transmission, and to those values obtained in [3H]L-glutamate binding assays. [3H]2-amino-4-phosphonobutyric acid does not exhibit significant binding to the Cl-/Ca2+-independent L-glutamate binding site(s), nor to the Na+-dependent L-glutamate binding site (up to 50 mM Na+). These data provide further evidence that the physiological action of 2-amino-4-phosphonobutyric acid is mediated by the previously described Cl-/Ca2+-dependent L-glutamate binding sites, and provides an assay system which is optimal for the study of these sites.

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus.

There is considerable evidence that pathways of the hippocampus use an excitatory amino acid as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.

Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes.

The pharmacology of recombinant NR1a/NR2D NMDA receptors expressed in Xenopus oocytes was examined and compared to the pharmacology of NR1a/NR2A, NR1a/NR2B and NR1a/NR2C heteromers. The NR1/NR2D heteromer showed a pharmacological profile distinct from each of the other NR1/NR2 heteromers. This unique pharmacological profile was characterized by a relatively lower affinity for the agonist homoquinolinate and the antagonists 2-amino-5-phosphonopentanoate (D-AP5) and (R,E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPPene) but not for the antagonists (+/-)-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid (PBPD) and alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid (EAB515). NR2D-containing receptors displayed a pharmacological profile most similar to that observed for receptors containing the genetically related NR2C subunit. These findings parallel observations obtained for native NMDA receptors in the medial thalamus (presumed to contain NR2D subunits) and forebrain (presumed to contain NR2A and NR2B subunits). Thus, only compounds that discriminate between either NR2A- or NR2B-containing heteromers and NR2D-containing heteromers also discriminate between forebrain and medial thalamic NMDA receptors. While the pharmacology of the NR1a/NR2D receptor shows many parallels to the medial thalamic NMDA receptor, some differences were observed. Certain compounds which discriminate between medial thalamic and cerebellar (presumed to contain NR2C subunits) receptors (e.g., homoquinolinate, D-CPPene) did not show a similar selectivity for NR1a/NR2D receptors relative to NR1/NR2C receptors. Co-expression of NR1a, NR2B and NR2D subunits in Xenopus oocytes resulted in the formation of heteromeric complexes with unique pharmacological properties, suggesting the co-existence of these two distinct NR2 subunits in the same receptor complex.