Effects of reboxetine and sertraline treatments alone and in combination on the binding properties of cortical NMDA and β1-adrenergic receptors in an animal model of depression

Summary. Changes to the binding properties of cortical N-methyl-D-aspartic acid (NMDA) and beta-adrenergic receptors have both been reported as potential indicators of antidepressant activity. In the present investigation we examined the effects of the noradrenaline reuptake inhibitor, reboxetine, the serotonin reuptake inhibitor, sertraline, alone and in combination on the binding properties of cortical NMDA receptors and cortical β₁-adrenoceptors following 14 days of treatment in the olfactory bulbectomized rat model of depression. A decrease in the potency of glycine to displace the strychnine insensitive glycine antagonist [³H] 5,7 dichlorokynurenic acid (5,7 DCKA) was observed in cortical homogenates of OB rats when compared to sham-operated controls. Similarly, treatment with the combination of reboxetine and sertraline for 14 days produced a decrease in the potency of glycine when compared to vehicle treated controls. By contrast neither olfactory bulbectomy or drug treatment significantly altered basal or glycine enhanced binding of the non-competitive NMDA antagonist [³H] MK-801 in cortical homogenates. Reboxetine alone, and in combination with sertraline, down-regulated [³H]-CGP 12177 (a selective β-adrenoceptor antagonist) binding in both OB and sham-operated animals. The lack of a bulbectomy effect in the [³H] CGP-12177 binding assay, and the fact that olfactory bulbectomy and antidepressant treatments produce a similar change to the potency of glycine at the NMDA receptor, suggests that these tests do not provide a neurochemical marker for either the behavioral hyperactivity deficit or antidepressant response in the model. Received February 6, 2000; accepted March 6, 2000     

Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: Ionic influences

Ammonium salts of some anions decrease the potency of glycine in inhibiting [³H]strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC₅₀ of glycine in inhibiting the [³H]strychnine binding, their capacity to reduce the [³H] strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of [³H]strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting [³H]strychnine is not. Binding of [³H]strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li⁺, K⁺, and Na⁺ up to 150 mM decrease [³H]strychnine binding, while higher concentrations of the cations increase [³H]strychnine binding. Inhibition by glycine of [³H]strychnine binding is enhanced by low concentrations of these cations.     

The glycine receptor deficiency of the mutant mouse spastic: evidence for normal glycine receptor structure and localization

Homozygotes of the mutant mouse spastic exhibit reduced binding of 3H-strychnine to homogenates from various regions of the CNS compared with unaffected littermates (White and Heller, 1982). Here we report evidence that the spastic mutation coincides with a reduced concentration and an unaltered structure of the glycine receptor in spinal cord. Scatchard analysis of 3H-strychnine binding revealed a single binding site with a Bmax of 267 +/- 62 fmol/mg protein for spastic and of 864 +/- 220 fmol/mg protein for control mice; no difference was found for the corresponding KD values. Also Ki values of glycine for 3H-strychnine binding and displacement of 3H-strychnine by beta-alanine and taurine were indistinguishable for both preparations. Photoaffinity labeling of synaptic membranes with 3H-strychnine identified an Mr = 48,000 polypeptide in both control and spastic mouse membranes. Tryptic digestion of these membranes produced radiolabeled peptide fragments of identical molecular weights, suggesting that the proteolytic cleavage sites around the antagonist binding site are conserved in the mutant glycine receptor protein. Glycine receptors from both control and mutant mice were purified by affinity chromatography on aminostrychnine agarose. SDS/PAGE revealed three polypeptides of Mr = 48,000, 58,000, and 93,000 in both receptor preparations. Monoclonal antibodies directed against different subunits of the glycine receptor were applied to an enzyme-linked immunosorbent assay. The same pattern of immunoreactivity was obtained for glycine receptor from spinal cord of spastic homozygotes, control mice, and rats, suggesting conservation of the antigenic epitopes in the mutant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy

Recent studies have shown that muscarinic receptors in brain hypoglossal nuclei exhibit a loss of specific ligand binding in response to axotomy of the hypoglossal nerve. The mouse hypoglossal nucleus contains a high level of receptors for the inhibitory neurotransmitter, glycine; the ligand [3H]strychnine binds to the glycine receptor with high affinity. In the present study [3H]strychnine binding in mouse hypoglossal nuclei was examined at 1 to 150 days after unilateral lesions of the hypoglossal nerve. Brains were sectioned on a cryostat, thaw-mounted onto microscope slides, incubated with [3H]strychnine and processed for light microscopic autoradiography. Receptor density was assessed by counting silver grains in photomicrographs of operated and control nuclei. During the first 25 days after axotomy grain density fell to 50 percent of that of the control nucleus. After this time grain density slowly increased, returning to control levels by 150 days post lesion. These data indicate that glycine receptors on the axotomized cells of the hypoglossal nucleus are lost when connection with the target muscles of the tongue is interrupted, and that the receptors reappear when the hypoglossal nerve regenerates. It is suggested that excitatory and inhibitory neurotransmitter receptor systems may be regulated in a coordinated fashion by the functional state of the motoneuron.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the rat

Glycine levels and receptor binding were measured in the medulla and spinal cord of 2-month, 10-month, and 24-month-old Fischer 344 rats. The behavioral response to the administration of the glycine antagonist, strychnine, was also evaluated in 2- and 24-month-old animals to investigate the relevance of these parameters to the susceptibility to seizures. Significant reductions in glycine in both the spinal cord and medulla occurred from 2 to 24 months of age. The glycine precursors, serine and threonine, were decreased only in the spinal cord. [3H]Strychnine binding was also decreased by 38% and 34% in the medulla and spinal cord, respectively, of 24-month-old rats compared to 2-month-olds. [3H]GABA binding was similarly reduced while no age-related changes in [3H]diazepam binding in the spinal cord were detected. Comparison of 2- and 24-month-old animals after systemic injection of 1.75 mg/kg strychnine showed that senescent animals have a higher incidence of seizures and mortality compared to young animals. Decreases in glycinergic neurotransmission may lower strychnine seizure threshold in the aged animal.     

Strychinine binding associated with synaptic glycine receptors in rat spinal cord membranes: ionic influences.

Ammonium salts of some anions decrease the potency of glycine in inhibiting (3H)strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC50 of glycine in inhibiting the (3H) strychnine binding, their capacity to reduce the (3H) strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of (3H)strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting (3H)strychnine is not. Binding of (3H)strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li+, K+, and Na+ up to 150mM decrease (3H)strychnine binding, while higher concentrations of the cations increase (3H)strychnine binding. Inhibition by glycine of (3H)strychnine binding is enhanced by low concentrations of these cations.     

Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system

Accumulation of [3H]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [3H]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [3H]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.     

Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.     

Developmental maturation of the N-methyl- -aspartic acid receptor channel complex in postnatal rat brain

The N-methyl- -aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [³H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors — an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1–day 14) is highly sensitive to spermidine, -glutamate alone potentiates [³H]MK-801 binding, and glycine failed to potentiate an -glutamate-induced increase in [³H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [³H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [³H]MK-801 binding was not seen in the presence of -glutamate alone, and -glutamate and glycine or -glutamate and spermidine or -glutamate, glycine and spermidine together, significantly increased [³H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.     

Solubilization of the glycine receptor from rat spinal cord

Glycine receptors, as detected by glycine-displaceable [3H]strychnine binding, were solubilized from a membrane fraction of rat spinal cord by the non-ionic detergent Triton X-100. The solubilized material retained its high affinity for [3H]strychnine and exhibited the typical pharmacological properties of the membrane-bound glycine receptor. On sucrose density gradients, the solubilized receptor had a sedimentation coefficient of 8.3 +/- 0.4 S. Gel exclusion chromatography on Sepharose 6 B in the presence of phosphatidylcholine gave a Stokes radius of 7.3 +/- 0.3 nm.     

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.     

Strychnine-insensitive [H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Ontogeny of glycine-enhanced [3H]MK-801 binding to N-methyl-D-aspartate receptor-coupled ion channels.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is thought to play a critical role in neuronal development, differentiation and plasticity. A number of studies have shown an enhanced sensitivity to NMDA receptor ligands in neonatal animals. This study examined the ontogenetic changes in the glycinergic modulation of NMDA-coupled cation channels in the developing central nervous system of rat pups. The nonequilibrium binding of the specific channel ligand [3H]MK-801 was used as a measure of NMDA channel access. Glycine (10(-5) M) enhancement of [3H]MK-801 binding at 2 h in forebrain membranes from adult rats was significantly greater than that observed in tissues from 8- to 28-day-old rat pups. This difference was due to changes in the efficacy, but not potency of glycine. The observed ontogenetic changes in the efficacy of glycine-enhanced [3H]MK-801 binding were attributable to developmental changes in receptor site density, as determined by equilibrium [3H]MK-801 saturation isotherms. Kinetic studies revealed that glycine increased the association rate constants of [3H]MK-801 in 8-day and adult membranes by a similar magnitude (0.111 +/- 0.021 vs 0.094 +/- 0.009 nM-1 h-1, respectively). Similarly, the fractional amount of [3H]MK-801 bound (i.e., amount bound at time t normalized to amount bound at equilibrium) in the presence of glycine was relatively constant throughout neonatal development. These findings suggest that the allosteric modulation of the NMDA ionophore by glycine is similar in postnatal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

TCP binding: A tool for studying NMDA receptor-mediated neurotransmission in kindling

Findings from numerous pharmacological and electrophysiological studies have uniquely implicated the N-methyl-D-aspartate (NMDA) receptor in kindling. Recent findings indicate that this receptor is regulated by ligands acting at both amino acid (NMDA and glycine) and ion (Zn++ and Mg++) binding sites. To examine the role of the NMDA receptor in kindling it will be necessary to understand how ligands for these different binding sites interact to control activation of the NMDA receptor. To this end we examined a biochemical tool for measuring opening of the NMDA receptor-gated ion channel (NMDA channel). [3H]N-(1-[thienyl] cyclohexyl)piperidine (TCP) binding to brain membranes is stimulated by NMDA and glycine receptor agonists. We have shown that NMDA and glycine increase TCP binding by increasing the access of TCP to its site. Moreover, the pharmacology of the NMDA and glycine binding sites regulating TCP binding is identical to that of the sites regulating NMDA evoked currents. These findings strongly suggest that glycine and NMDA regulate TCP binding by increasing the opening of the NMDA channel. That is NMDA and glycine increase the overall time that the channel is open thereby increasing the time available for TCP to diffuse to its binding site. These findings support the use of TCP binding (association rate) as a marker of channel opening and thereby permit measurement of NMDA receptor activation and ligand binding under identical conditions. This will allow direct testing the hypothesis that an alteration in the NMDA receptor/channel complex itself underlies the increased seizure response of kindled animals.     

Putative glycine receptors in Hydra: a biochemical and behavioural study

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.     

Localization of high affinity [3H]glycine transport sites in the cerebellar cortex

A study was made of [3H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [3H]glycine at the low concentration used. Glial cells also took up [3H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [3H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [3H]GABA uptake (Ki over 600 microM vs the Kt of about 20 microM) and that GABA is an even weaker inhibitor of [3H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [3H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [3H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

The quaking mouse: an epileptic mutant with alterations affecting the modulatory mechanisms of the NMDA receptor complex

The binding of [³H]glutamate and of [³H]1-(1-(2-thienyl)cyclohexyl)piperidine ([³H]TCP) has been examined in the genetically epileptic mutant mouse, quaking. The density of [³H]glutamate binding sites did not differ between the quaking mice and their controls of the same strain. In the absence of exogenous glutamate or glycine, the density of [³H]TCP binding sites was also similar in the two strains. In both the mutants and their controls, exogenously added glutamate, glycine and glutamate plus glycine dose-dependently increased the binding of [³H]TCP. In the 3 conditions, the modulation of [³H]TCP binding was significantly more efficient in the quaking mice than in the controls. Furthermore, in the presence of glutamate (10⁻⁵ M), the increase of the affinity of the ligand for the ion channel binding site was higher in the mutants than in the controls. These results suggest that the modulatory mechanisms of the

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receptor complex might be altered in these mutants. These alterations might be related to the previously observed anticonvulsant properties of NMDA receptor antagonists in the quaking mouse model of inherited epilepsy.     

Effect of cocaine and cocaine congeners on veratridine-induced depolarization in mouse cerebrocortical synaptoneurosomes

Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-alpha-benzoate binding in mouse brain synaptosomes.     

Kinetic mechanisms of glycine requirement for N-methyl-D-aspartate channel activation.

Glycine potentiates N-methyl-D-aspartate (NMDA) receptor-mediated responses via its interaction with a strychnine-insensitive glycine recognition site. We have previously shown that the potent glycine receptor antagonist 7-chlorokynurenic acid (7Cl-KYN) dose-dependently inhibits [3H]MK-801 binding to the PCP receptor and that this effect is reversed by glycine. [3H]MK-801 binding to the PCP receptor within the NMDA receptor-gated ion channel is a measure of channel activation. Association of PCP receptor ligands is biexponential with the fast component of binding serving as a marker of activated NMDA channels. In the present study we utilize 7Cl-KYN as a probe of the kinetic mechanism of the glycine effect upon NMDA receptor functioning. In the presence of L-glutamate, incubation with 7Cl-KYN completely abolished the fast component of [3H]MK-801 association in 4 out of 5 experiments. In the fifth experiment where the fast component was detected, it accounted for less than half of that seen in the presence of L-glutamate alone. 7Cl-KYN-induced inhibition of the fast component of [3H]MK-801 association was reversed by the addition of glycine. Since the fast component represents ligand binding to the PCP receptor via the open NMDA channel, selective reduction of this component by 7Cl-KYN indicates that glycine receptor antagonists reduce the probability of channel opening, and also that the selective reduction in the component of [3H]MK-801 binding that manifests fast kinetics can serve as a marker for glycine antagonists.