Daniel T. Monaghan

Anatomical organization of excitatory amino acid receptors and their pathways

Abstract Synapses that employ excitatory amino acids (EAA) as their neurotransmitter use multiple combinations of receptors, apparently yielding different functional properties. The most well-characterized receptor class, the N-methyl-d-aspartate (NMDA) receptor, is found throughout the brain but primarily in telencephalic regions. Phencyclidine and glycine binding sites, which interact allosterically with NMDA receptors, have almost identical distributions. NMDA sites also extensively overlap with the quisqualate excitatory amino acid receptor class, but are less often found co-localized with the kainate receptor class. Evidence obtained with presynaptic markers - high affinity glutamate or aspartate uptake, Ca 2+ -dependent release, and glutamate and aspartate contents - each indicate that EAA are major transmitters of corticocortical, corticofugal, and sensory systems. Recent advances in the histological analysis of these markers are now providing a more detailed map of the excitatory amino acid system and this anatomical map appears to correspond to the distribution of the sum of the receptors. Thus the receptor systems may represent distinct, anatomically-organized, subsystems of excitatory amino acid-mediated neurotransmission.

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.

Trans-ACPD, a selective agonist of the phosphoinositide-coupled excitatory amino acid receptor

Excitatory amino acids (EAA) are thought to mediate their actions through at least five receptor subtypes. Four of these types appear to gate ion channels and have been named for agonists by which they are selectively activated: N-methyl-Daspartate (NMDA), kainate, a-amino-3-hydroxy5-methyl isoxazole-4-propionlc acid (AMPA), quisqualate and 2-amino-4-phosphonobutyrate (AP4). There is now compelling evidence from studies of phospholnositlde (PI) metabolism indicating that there is a fifth distinct EAA receptor class (for references see Monaghan et al., 1989). In both mRNA-injected oocytes and rat Inppocampal slices PI metabolism is activated by Lglutamate, ibotenate and quisqualate. This appears to represent a distinct receptor class because tins site is not activated by NMDA, kainate, AMPA or L-AP4, nor is it blocked by their antagonists. To date, pharmacological analysis of the PIcoupled EAA receptor has been hampered by the absence of a selective agonist. This presents a particular problem for the characterization of this receptor because simultaneous activation of the other EAA receptors (NMDA, kainate or AMPA) along with the PI-coupled receptor can inhibit subsequent PI metabohsm (Palmer et al., 1988). In this study, we report that the glutamate analogue

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.

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.

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (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.

Glutamate receptors and phosphoinositide metabolism: stimulation via quisqualate receptors is inhibited by 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.

Localization of N-acetylaspartylglutamate-like immunoreactivity in selected areas of the rat brain

N-acetylaspartylglutamate (NAAG) was detected immunohistochemically in the rat brain using an antiserum which recognizes carbodiimide-fixed NAAG. NAAG-like immunoreactivity is described in 5 areas of the brain; olfactory bulb, septal nuclear area, lateral geniculate nucleus, superior colliculus and the entorhinal cortex/hippocampal formation. Mitral cells of the olfactory bulb and neurons concentrated in the medial septum were densely immunostained. A dense population of immunoreactive puncta was found in the superior colliculus and lateral geniculate nucleus (LGN). The LGN also contained immunoreactive neurons. The entorhinal cortex contained numerous immunoreactive cells in layers II-III while the hippocampus had few neurons that were NAAG-positive.

[3H]TCP binding sites in Alzheimer's disease

Quantitative autoradiography was used to determine the density and distribution of [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine ([3H]TCP) binding sites in human hippocampal tissue sections from control and Alzheimers disease patients. Some Alzheimers cases showed no changes in binding site density while other cases showed substantial declines in the CA1 region. [3H]TCP binding in the CA1 region from Alzheimers patients was reduced an average of 40% while the other hippocampal regions were unaffected. It is proposed that the loss of [3H]TCP sites in the hippocampal CA1 region of certain Alzheimers cases is associated with the greater cell loss observed in cases of severe Alzheimers disease.

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.