N -methyl -D -aspartate binding sites in the vertebrate central nervous system: Characterization using quantitative autoradiography of tritium MK-801 binding.

Excitatory amino acids (EAAs) such as glutamate or aspartate, exert their effects in vertebrate central nervous system (CNS) through at least four receptor subtypes. One of these, the N-methyl-D-aspartate (NMDA) receptor, may play an important role in learning, memory, motor function and synaptic plasticity. A quantitative autoradiographic assay using ($\sp3$H) MK-801, which binds in the NMDA receptor ion channel, was developed to study the modulation and pharmacology of the NMDA receptor in vertebrate brain. ($\sp3$H) MK-801 binding sites were distributed heterogenously throughout rat brain and appeared to label the NMDA receptor. Glutamate and glycine enhanced ($\sp3$H) MK-801 binding while competitive NMDA antagonists, glycine antagonists and zinc inhibited ($\sp3$H) MK-801 binding allosterically. ($\sp3$H) MK-801 binding could thus be used in a quantitative autoradiographic assay as an indicator of NMDA receptor activation. Differential activation of the NMDA receptor complex revealed the ($\sp3$H) MK-801 binding could be used to distinguish pharmacologically and regionally distinct subtypes of the NMDA receptor in rat CNS. The regionally distinct NMDA receptor subtypes were defined by differential regulation at the NMDA recognition site and the strychnine-insensitive glycine site. At least three NMDA receptor subtypes were distinguished by CPP, a competitive NMDA antagonist, inhibition of ($\sp3$H) MK-801 binding. Inhibition of ($\sp3$H) MK-801 binding by 7-chlorokynurenic acid, a glycine antagonist, defined two additional regionally specific NMDA receptors. The phylogeny of EAA binding sites was investigated in turtle and pigeon brain. In turtle forebrain, non-NMDA and NMDA receptors were distributed heterogenously and the pharmacology of ($\sp3$H) MK-801 binding was similar to rat and pigeon. In pigeon forebrain sections, NMDA receptors were distributed heterogenously and ($\sp3$H) MK-801 binding was regulated by NMDA and glycine antagonists. In pigeon hindbrain sections, a unique profile of binding to NMDA, glycine and MK-801 sites in the optic tectum strongly suggested the presence of NMDA receptor heterogeneity. These studies suggest that ($\sp3$H) MK-801 binding can be used to measure NMDA receptor regulation. In rat brain there are different subtypes of NMDA receptors based on their differential pharmacology. Regulation of NMDA receptors in pigeon and turtle forebrain is similar to rat; furthermore, NMDA receptors in pigeon hindbrain are heterogenous. Thus, subtypes of the NMDA receptor are present in vertebrate CNS and they vary regionally and across species.Ph.D.Biological SciencesHealth and Environmental SciencesNeurosciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128836/2/9208646.pd