NMDA Receptor Density Research Articles

Loss of [3H]MK801 binding sites in brain in congenital ornithine transcarbamylase deficiency.

Alterations of excitatory amino acid neurotransmitters have previously been described in brain in congenital ornithine transcarbamylase (OTC) deficiency. In order to further elucidate the role of the glutamatergic neurotransmitter system in OTC deficiency, densities of binding sites for [3H]MK801, an NMDA receptor antagonist ligand were measured by quantitative receptor autoradiography in the brains of chronically hyperammonemic sparse-fur mice (spf), mutant mice with a congenital defect of OTC. [3H]MK801 binding site densities were significantly reduced by up to 57% (p < 0.01) in 16 out of 17 brain regions of OTC-deficient mice. Such changes could result from either neuronal cell loss in these animals or from "down-regulation" of these sites as a consequence of exposure to increased extracellular concentrations of glutamate or quinolinic acid, two known endogenous NMDA receptor ligands previously found to be increased in brain in chronic hyperammonemic syndromes. Reduced NMDA receptor densities in congenital OTC deficiency could represent an adaptive mechanism of protection against further excitotoxic brain injury.

A photoaffinity probe for the polyamine site regulating the NMDA receptor

Polyamines produce on the NMDA-receptor channel activity a regulatory effect subsequent to their binding to specific sites on the receptor-complex. The photoactivatable polyamine derivative L-azidophenylspermine shows properties which suggest its potential as a photoaffinity probe to investigate the nature and topology of these sites. In the dark, its effect on the binding of tritiated N-(1-[thienyl] cyclohexyl)piperidine ([ 3H]TCP) to synaptosomal plasma membranes is similar to that of diaminodecane. Arcaine antagonizes both the effects of L-azidophenylspermine and diaminodecane on [ 3H]TCP binding. L-Azidophenylspermine competes in post-synaptic densities with tritiated spermidine for a unique binding site with an EC 50 similar to that of spermine. Upon irradiation, L-azidophenylspermine incorporates into this material with a high efficiency to a level consistent with both the B max for tritiated spermidine and the estimated density of NMDA receptors in this fraction.

D-Cycloserine, a partial agonist at the glycine site, enhances the excitability of dentate granule cells in vivo in rats.

The present study investigated the dose-dependent effects of d-cycloserine, a partial agonist at the glycine modulatory site associated with the NMDA receptor complex, on the hippocampal field potentials of dentate granule cells in awake, freely moving rats. Five sequential field potentials were recorded from the dentate gyrus of the dorsal hippocampus, by stimulating the perforant path in the entorhinal cortex at 30-s intervals. The slope of the population excitatory postsynaptic potential (e.p.s.p.) and the amplitude of the population spike of these field potentials were analysed and averaged with a computer. The effects of d-cycloserine (1.0, 3.0, 9.0 mg/kg) were recorded 40 min and 24 h after the i.p. injection. Although the slope of the population e.p.s.p. showed no significant change after the administration of d-cycloserine, the high doses produced a substantial increase in the amplitude of the population spike. This increase was observed 40 min but not 24 h after the injection. These findings indicate that d-cycloserine does not change the synaptic input from the perforant path to the granule cells but dose dependently enhances the excitability of the hippocampal dentate granule cells. In addition, the data give further support to the suggestion that in the brain area where NMDA receptor density is relatively high, the glycine site of the NMDA receptor may not be fully saturated by endogenous glycine in normal in vivo conditions. This suggests that there is a possibility for pharmacological modulation of NMDA receptor-mediated synaptic events by exogenous glycine or glycine analogues.

Increased hypothalamic glutamate receptors induced by water deprivation.

To examine the role of receptor changes in the adaptive response to physiological stimulation, the density and distribution of excitatory amino acid receptors within the hypothalamus and other brain regions were examined in rats deprived of water for 2 days. Membrane binding assay revealed an increase in glutamate receptor density and a small shift in the affinity of glutamate for the receptor. Regional analysis of these changes by receptor autoradiography specific for NMDA, non-NMDA or metabotropic glutamate receptor binding indicated that NMDA and metabotropic receptor densities are increased in the brain. Regional increases were found principally for the NMDA receptor binding within the supraoptic nucleus, anterior hypothalamus, caudate-putamen and globus pallidus with no significant changes in 24 other brain regions. No significant changes were found in any brain regions for AMPA receptors. Metabotropic and kainate receptors tended to parallel the NMDA receptor changes, although few regions reached statistical significance. These changes indicate that brain regions associated with water balance regulation show selective adaptive increases in NMDA receptors during water deprivation which may facilitate prolonged activation of these cells.

Autoradiographic localisation of NMDA binding sites in brainstem cardiorespiratory areas of adult and newborn cats

Quantitative autoradiography was used to determine the distribution of [ 3H]1-[1-(2-thienyl)cyclohexyl] piperidine ([ 3H]TCP) binding sites in the brainstem in order to localize the possible targets activated by excitatory amino acids in adult and newborn cats during autonomic rhythmic functions. Medium to high densities of binding sites were found in the nucleus tractus solitarius complex and the laterodorsal part of the pontine tegmentum. In the kitten, there was an apparent higher density in cranial motor nuclei proximal to these structures. It is concluded that there is an overlapping between the high density of NMDA receptors and the localization of cardiorespiratory neurons in cat as well as in kitten.

The role of NMDA receptors in information processing.

In this review, we have concentrated on the parallels between the cellular properties of the NMDA receptor and a variety of functional properties within sensory and motor systems. Of course, the NMDA channel exists within the cell in conjunction with a variety of other channels, including non-NMDA channels. Although the NMDA receptor is unique in a cellular sense--it is the only ligand-gated channel that is also voltage dependent and calcium permeable--it is not unique in a functional sense. A cell that has non-NMDA receptors and voltage-sensitive channels will also exhibit nonlinear behavior. Moreover, Buhrle & Sonnhof (1983) demonstrated some time ago that calcium flows into frog motor neurons through more than one type of calcium channel. The contribution to the inflow of calcium from NMDA channels may vary from cell to cell and could easily be a minor proportion of the total. Many authors have pointed out that the NMDA channel has a low conductance at a resting potential of -70 mV. However, many cells in the nervous system are depolarized from -70 mV by excitatory input. Thus, as pointed out above. NMDA receptors make a contribution to the tonic or spontaneous activity of cells in both visual cortex and spinal cord. In practice, many cells are probably working in a range of membrane potentials where the NMDA channels are always open to some extent. Even in the hippocampal slice where a substantial amount of afferent input is removed, NMDA receptors contribute to spontaneous activity (Sah et al 1989). Does the NMDA receptor act as a switch? Does it act as an AND gate? The suggestion that it may act as a switch comes from work on LTP in the hippocampus, which is readily produced by high-frequency stimulation and is abolished by APV. However, activation of the NMDA receptor is only the first in a sequence of reactions leading to LTP: In theory, switch-like behavior could also be produced by calcium-buffering systems within dendritic spines, or by enzymatic processes (Lisman 1985; Zador et al 1990). Fox & Daw (1992) have modeled the action of NMDA and non-NMDA receptors that are activated in parallel with each other, and shown that the occurrence of switch-like behavior depends on the relative density of NMDA versus non-NMDA receptors. Switch-like behavior is not seen in the visual cortex, but might be seen in the hippocampus if the relative density of NMDA receptors there was higher than in the visual cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures

Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 μ M), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 μ M) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 m M) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.

Increase inω 3 (peripheral type benzodiazepine) binding sites in the rat cortex and striatum after local injection of interleukin-1, tumour necrosis factor-α and lipopolysaccharide

The possible involvement of lymphokines in the glial reaction/proliferation that follows brain injury has been investigated by measuring the density ofω 3 (peripheral type benzodiazepine) binding sites associated to glial cells and macrophages after local injection of lymphokines in the rat cerebral cortex and striatum.ω 3 Site densities were measured either by quantitative autoradiography in brain sections or by conventional binding in membrane using [ 3H]PK 14105 or [ 3H]PK 11195 as ligands. Intracortical or intrastriatal infusion of interleukin-1 (10 and 20 units) caused a marked increase in the density ofω 3 sites (+83% and +80%, respectively, when compared to saline-infused animals) around the injection site at 7 days postinjection. There was a good spatial correspondence between the autoradiographic distribution ofω 3 sites and the distribution of reactive astrocytes (as assessed by GFAP immunostaining) or acid phosphatase rich cells (phagocytes). Significant increases inω 3 site densities were also observed in striatal homogenates 1 week after local administration of tumor necrosis factor-α (TNF-α). The maximal increase (+80%) was observed after the administration of 3 units, higher and lower doses resulting in smaller increases. Intrastriatal injection of E. coli lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate interleukin-1 and TNF-α production by microglial cells in culture, also resulted in significant increases inω 3 site densities in striatal homogenates (maximal increase, +170% 1 week after the injection of 200 ng). In contrast, no change in the density of two binding sites associated with neurons ([ 3H]TCP which indexes NMDA receptor density and [ 3H]Ro15 1788 which binds to centralω 1–2 (benzodiazepine) receptors) was observed after intrastriatal injection of interleukin-1, TNF-α or LPS. The intrastriatal injection of interleukin-2 (5–20 units) or of the chemotactic peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP) failed to alterω 3 site or NMDA receptor densities in striatal homogenates. Based on the present results, we suggest that the increase inω 3 site densities observed in experimental lesions and in human neuropathological states may be mediated by a sequential mechanism involving the activation of microglia or monocytes, the release of interleukin-1 and/or TNF-α and the promotion, by these cytokines, of the astroglial reaction.

A quantitative study of the actions of excitatory amino acids and antagonists in rat hippocampal slices.

1. A quantitative pharmacological investigation of the actions of excitatory amino acids on hippocampal CA1 neurones has been made using a new slice preparation developed for grease gap recording; d.c. potential was measured across a grease barrier placed between alvear fibres and the bathing medium. 2. In Mg2+-free perfusate, N-methyl-D-aspartate (NMDA, 1-100 microM), quisqualate (1-500 microM), kainate (1-200 microM) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1-100 microM) caused dose-dependent depolarizations. 3. The dose-response relationships were fitted to logistic expressions. The maximum responses to AMPA, NMDA and kainate were similar; their respective EC50 values were 5, 13 and 23 microM. Quisqualate had a smaller maximum; its EC50 value was 10 microM. The slopes of the dose-response relationships were different for the 4 agonists; the order of steepness of the slopes was NMDA greater than AMPA greater than kainate greater than quisqualate. 4. Similar amino acid-induced depolarizations were observed in slices of just the CA1 region or in whole slices bathed in tetrodotoxin. Isolated alvear fibres, however, were insensitive to the excitatory amino acids. 5. D-2-Amino-5-phosphonovalerate (APV, 50 microM) selectively and reversibly antagonized responses induced by NMDA (apparent pA2 = 5.21). 6. Kynurenic acid (1 mM) reversibly depressed responses to the three agonists tested. The dose-ratios for antagonism of AMPA, kainate and quisqualate were 6.9, 5.6 and 4.6 respectively. 7. This preparation has a different sensitivity profile to agonists from those of previously reported preparations of spinal cord, neocortex and cerebellum. The greater sensitivity to NMDA may be due to the higher density of NMDA receptors in the hippocampus. The effects of the antagonists, APV and kynurenate, are similar to those found in other brain areas.