Involvement of AMPA Receptors in Maintenance of Memory for a Passive Avoidance Task in Day-old Domestic Chicks (Gallus domesticus)

Day-old chicks (Gallus domesticus) were trained on a one-trial passive avoidance task where the aversive stimulus was an unpleasant tasting substance, methyl anthranilate. Chicks were killed 6.5 h after training. The kinetic parameters of [³H] α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) binding were determined using quantitative receptor autoradiography and Scatchard analyses in 15 discrete forebrain regions of trained and control (water-trained) chicks, revealing two components of binding in each. K_D values showed some regional variation, but were 22.2 ± 1.1 nmol l^-1 for the high-affinity component and 685 ± 25 nmol l^-1 for the low-affinity component of binding to whole forebrain sections from control chicks. Analyses also revealed that Hill coefficients were significantly less than 1 in all regions measured. A significant decrease in K_D for the low-affinity component occurred bilaterally in the intermediate andmedial hyperstriatum ventrale (IMHV; left, 34.8%; right, 33.3%), a region that has previously been shown to be implicated in the processes of memory formation, following passive avoidance training. A significant decrease in K_D for the high-affinity component occurred in the right palaeostriatum augmentatum (19.5%). Significant decreases in B_max accompanied the K_D alterations in both cases. Additionally, bilateral intracerebral injections (administered 4.5-5.5 h after training) into the IMHV of 500 nmol l^-1 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA glutamate receptors (particularly AMPA receptors), resulted in amnesia for one-trial passive avoidance training in day-old chicks tested 6.5 h after training. Unilateral injections administered 5.5 h after training had a significant but smaller effect. Injections administered before training or 5 min after training had no effect on learning or retention of memory for the task. These results show that the affinity of AMPA receptors increases at a time point after passive avoidance training at which their inhibition by CNQX blocks maintenance of long-term memory for this task.     

Non-NMDA excitatory amino acid receptors in the ventral tegmental area mediate systemic dizocilpine (MK-801) induced hyperlocomotion and dopamine release in the nucleus accumbens

This study investigated the putative role of non-NMDA excitatory amino acid (EAA) receptors in the ventral tegmental area (VTA) for the increase in dopamine (DA) release in the nucleus accumbens (NAC) and behavioral stimulation induced by systemically administered dizocilpine (MK-801). Microdialysis was utilized in freely moving rats implanted with probes in the VTA and NAC. Dialysates from the NAC were analyzed with high-performance liquid chromatography for DA and its metabolites. The VTA was perfused with the AMPA and kainate receptor antagonist CNQX (0.3 or 1 mM) or vehicle. Forty min after onset of CNQX or vehicle perfusion of the VTA, MK-801 (0.1 mg/kg) was injected subcutaneously. Subsequently, typical MK-801 induced behaviors were also assessed in the same animals by direct observation. MK-801 induced hyperlocomotion was associated with a 50% increase of DA levels in NAC dialysates. Both the MK-801 evoked hyperlocomotion and DA release in the NAC was antagonized by CNQX perfusion of the VTA in a concentration-dependent manner. None of the other rated MK-801 evoked behaviors, e.g. head weaving or sniffing, were affected by CNQX perfusion of the VTA. By itself the CNQX or vehicle perfusion of the VTA alone did not affect DA levels in NAC or any of the rated behaviors. These results indicate that MK-801 induced hyperlocomotion and DA release in the NAC are largely elicited within the VTA via activation of non-NMDA EAA receptors, tentatively caused by increased EAA release. Thus, the locomotor stimulation induced by psychotomimetic NMDA receptor antagonists may not only reflect impaired NMDA receptor function, but also enhanced AMPA and/or kainate receptor activation in brain, e.g., in the VTA. In view of their capacity to largely antagonize the behavioral stimulation induced by psychotomimetic drugs, such as MK-801, AMPA, and/or kainate receptor antagonists may possess antipsychotic efficacy. J. Neurosci. Res. 51:583–592, 1998. © 1998 Wiley-Liss, Inc.     

NMDA receptor antagonists inhibit apomorphine-induced climbing behavior not only in intact mice but also in reserpine-treated mice

The present study showed that the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 {(+)-5-methyl-10,11-dihydroxy-5H-dibenzo-[a,d]-cyclohepten-5,10-im ine hydrogen maleate}, ketamine, dextrorphan and dextromethorphan attenuated apomorphine-induced climbing behavior in reserpine-treated mice. In addition, the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid (AP-5) and D(-)-3-(2-carboxypipera-zine-4-yl)-propyl-1-phosphonic acid (CPP), also inhibited the apomorphine-induced climbing behavior in reserpine-treated mice as well as in intact mice. Previous work in our laboratory had shown that the noncompetitive NMDA receptor antagonists, MK-801, ketamine, dextrorphan and dextromethorphan cause a pronounced inhibition of apomorphine-induced cage climbing behavior in intact mice, suggesting the involvement of NMDA receptors in the glutamatergic modulation of dopaminergic function at the postsynaptic dopamine (DA) receptors. Therefore, the present results strongly support our previous conclusion that the NMDA receptors play important roles in the glutamatergic modulation of dopaminergic function at the postsynaptic DA receptors.     

Intranigral but not intrastriatal microinjection of the NMDA antagonist MK-801 induces contralateral circling in the 6-OHDA rat model

When systemically administered to unilaterally 6-OHDA lesioned rats, the non-competitive antagonists of (NMDA) receptors MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) produces a marked rotation response toward the lesioned side. In addition, the same type of injection in non-denervated rats increases locomotor activity without direction predominance. These findings suggest that MK-801 interacts with the dopaminergic pathway to induce motor activity. However, intracerebral injection of MK-801 (5 μg/0.5 μl) in the substantia nigra pars reticula a (SNr), either on the lesioned side or the intact side, induces a significant circling response, contraversive to the injection side. A similar injection of MK-801 (5 μg/1.0 μl) in the striatum causes no rotation response. These results show that the non-competitive NMDA antagonist can induce motor activity in the presence or in the absence of the dopaminergic pathway and that in the latter case its actions is exerted predominantly at the level of the substantia nigra, perhaps through suppression of the excitatory subthalamic drive.     

Adaptations of NMDA and dopamine D2, but not of muscarinic receptors following 14 days administration of uncompetitive NMDA receptor antagonists

Summary. Behavioral changes have previously been reported following administrations of uncompetitive NMDA receptor antagonists memantine, amantadine and MK-801 for 14 days, at the doses that produce plasma levels comparable to those seen in patients (20, 100 and 0.31 mg/kg/day respectively). Using the same doses, the effect on receptor binding (autoradiography) was studied in rats. [³H]MK-801 binding was increased in the dentate gyrus and CA3 region of the hippocampus (35.2 and 24.3% respectively) following 3 days S.C. infusion of memantine by ALZET minipumps. One daily injection of memantine for 14 days, increased [³H]MK-801 binding in the frontal cortex by 40.3%. The same treatment with amantadine did increase [³H]raclopride binding to dopamine D₂ receptors by 13.5%. None of these treatments changed the expression of muscarinic receptors. It is concluded that subchronic blockade of the NMDA receptor by uncompetitive antagonists at moderate (therapeutically-relevant) doses induced only minor changes in NMDA and dopamine D₂ receptor expression. Received September 18, 1998; accepted November 16, 1998     

Interaction between M1-muscarinic and glutamatergic NMDA receptors on an inhibitory avoidance task

It has been demonstrated that MK-801 potentiates the effects of the non-selective muscarinic antagonist scopolamine on memory in rats. In this study, we investigated the role of the M1-muscarinic receptor in this interaction, by administering different doses of dicyclomine (DIC) and MK-801 in combination to male Wistar rats before training on the inhibitory avoidance task. MK-801 and DIC in sub-effective doses were administered in combination. It was observed that MK-801 at a dose of 0.1125 mg/kg with a sub-effective dose of 8 mg/kg of DIC significantly impaired the retention test when compared with saline-treated animals, i.e. MK-801 potentiated the effects of dicyclomine on memory impairment. Our results suggest an important role for the M1-muscarinic receptor in the synergistic interaction between cholinergic muscarinic and glutamatergic NMDA receptors, which is in line with the findings that the interactive modulation between these two neurotransmitters systems constitutes an important mechanism in cognitive functions.     

The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets

The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [³H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na⁺,K⁺-ATPase activity was determined in a brain cell membrane preparation. [³H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5–50 nM [³H]MK-801 in the presence of 100 μM glutamate and glycine; and (2) binding assays at 10 nM [³H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 μM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na⁺,K⁺-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (B_max), but the apparent affinity was significantly increased, as indicated by a decrease in the K_d (dissociation constant) from the control value of 8.1 ± 1.6 nM to 5.5 ± 2.1 nM (P < 0.05). Augmentation of [³H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals. These data suggest that acute hypoglycemia may enhance the excitotoxic effects of glutamate in the newborn.     

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.     

Positive and negative modulation by AMPA- and kainate-receptors of striatal kainate injection-induced neuronal loss in rat forebrain

We investigated the roles of ionotropic glutamate receptor subtypes in mediating striatal kainate injection-induced neuronal loss in rat forebrain, using subtype-specific antagonists and histochemical staining. Our study demonstrates that kainate injected unilaterally into the striatum induces a massive neuronal loss in the rat ipsilateral forebrain through activation of kainate receptors and, to a limited extent, a consequent involvement of M-methyl-D-aspartate (NMDA) receptors, whereas activation of alpha-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA) receptors shows a neuroprotective effect. These and previous results suggest that three subtypes of ionotropic glutamate receptors play differential roles in mediating excitatory amino acid (EAA)-induced neurodegeneration.     

Glycoprotein Synthesis Is Necessary for Memory of Sickness-Induced Learning in Chicks

Can current biochemical models of memory account for sickness-induced learning? We show that chicks can form an association between pecking a coloured but tasteless lure and becoming ill (LiCI, i.p.) 30 min later. We go on to demonstrate amnesia for this association, induced by intracranial administration of 2-deoxygalactose (10 μmole per hemisphere, in a 10 γl vol), an inhibitor of the synthesis of glycoproteins of the synaptic membrane, 10 min before pecking. Further, we show that this 2-deoxygalactose-induced amnesia is not state dependent. Thus the brain representation of the lure must be held, and require macromolecular syntheses, similar to those found in other forms of learning, for a considerable time before it can be associated with new significant experience. This is incompatible with contiguous synaptic firing views of memory.     

Non-NMDA mechanism in the inhibition of cellular apoptosis and memory impairment induced by repeated ischemia in rats

The spatial memory impairment and expression of apoptotic cells in hippocampal CA1 cells were investigated in rats using single and repeated ischemia models. The neuroprotective and memory-improving effect of YM-90K, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, was compared to MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist. Twice-repeated ischemia, but not single ischemia, impaired the spatial memory and increased expression of apoptotic cells. YM-90K, given before and 6 h after the second reperfusion, significantly improved the memory and reduced the apoptotic cells 7 days after the second reperfusion in repeated ischemia. MK-801 neither improved the spatial memory nor reduced apoptotic cells. The present study showed that delayed expression of apoptotic cells is mediated by mechanisms involving AMPA receptors, but not by NMDA receptor, during the late phase after reperfusion. YM-90K could provide neuroprotective activity and improve the spatial memory impaired by repeated ischemia.     

Effects of intra-amygdala injections of NMDA receptor antagonists on acquisition and retention of inhibitory avoidance

These experiments examined the effects of intra-amygdala injections of NMDA receptor antagonists on the acquisition and retention of inhibitory avoidance. In Expt. I, rats received bilateral intra-amygdala injections of the NMDA antagonists D,L-AP5 (1-10 micrograms), D-AP5 (0.03-1 micrograms), CPP (0.125 or 0.375 microgram), or MK-801 (0.2 or 0.5 microgram) prior to training in a continuous multiple-trial inhibitory avoidance (CMIA) task. Acquisition of the task was not significantly affected by any of the drug injections. In contrast, all three competitive antagonists, D,L-AP5, D-AP5 and CPP, produced dose-dependent impairment of 48 h retention performance. Although the MK-801 injections did not significantly impair retention performance, the retention scores of the 0.5 microgram MK-801 group were bimodally distributed, indicating retention impairment in a subgroup of the animals given that dose. Intra-amygdala injections of 3 or 10 micrograms D,L-AP5 did not affect footshock sensitivity (Expt. II) or locomotor activity (Expt. III) and their retention-impairing effects were not due to induction of state dependency (Expt. IV). The retention-impairing effects of intra-amygdala injections of NMDA antagonists were not due to diffusion of the drugs dorsally: injections of 1 microgram D-AP5 into the striatal area directly above the amygdala impaired acquisition but not retention performance (Expt. V). The retention-impairing effects of 1 microgram D-AP5 or 0.5 microgram MK-801 were attenuated by giving additional training to the animals shortly after receiving intra-amygdala injections (Expt. VI). The implications of these findings for hypotheses concerning amygdala function in learning and memory are discussed.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Effect of ionotropic glutamate receptor antagonists on Fos-like immunoreactivity in the dorsal horn following transection of the rat sciatic nerve

Fos-like immunoreactivity (FLI) was investigated in the lumbar dorsal horn 2 h after transection of the rat sciatic nerve and sham operation. FLI following nerve transection was distributed through the medio-lateral extension of the superficial layer of the dorsal horn, while FLI after sham operation, tissue injury, was restricted to the lateral one-third of this layer. The number of FLI neurons in the lateral one-third was similar in the two operations, indicating that neurons expressing FLI in the medial two-thirds and in the lateral one-third of the superficial layer after nerve transection are derived from nerve injury and tissue injury, respectively. FLI in the lateral one-third, but not the medial two-thirds, after nerve transection was significantly reduced by pretreatment with NMDA and AMPA/KA receptor antagonists, indicating that there is a considerable difference in the contributions of ionotropic glutamate receptors to FLI in this layer induced by nerve injury and tissue injury.     

The Glutamate Transport Inhibitor L-trans-pyrrolidine-2,4-dicarboxylate Indirectly Evokes NMDA Receptor Mediated Neurotoxicity in Rat Cortical Cultures

Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4- dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC₅₀ in these cultures of 320 ± 157 μM. In astrocyte-poor cultures, an EC₅₀ for PDC of 50 ± 5 μM was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [³H]-D-aspartate, first we demonstrated that PDC caused significant efflux of [³H]-D-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC₅₀ of 18 ± 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [³H]-D-aspartate release. PDC-evoked release of [³H]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.     

MCPG Antagonizes Metabotropic Glutamate Receptors but not Long-term Potentiation in the Hippocampus

In the CA1 and CA3 regions of the guinea pig hippocampus, we have tested the ability of the new antagonist (RS) -α-methyl-4-carboxyphenylglycine (MCPG) to inhibit the well-known effects of ( trans )-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), a specific agonist of glutamate metabotropic receptors. Whole-cell recordings showed that MCPG was able to antagonize the blocking action of ACPD on I_AHP in the CA1 region. In addition, we report here that MCPG also antagonized the presynaptic inhibitory actions of ACPD on field excitatory postsynaptic potentials in both areas CA1 and CA3. Thus, MCPG proved to be an effective tool for determining physiological roles of the glutamate metabotropic receptors in synaptic transmission in the hippocampus. We next tested the possible effects of this antagonist on long-term potentiation (LTP). In completely blind experiments MCPG was without effect on LTP in both areas CA1 and CA3. In conclusion, our results suggest that, although MCPG is a valuable antagonist of the ACPD-sensitive receptors, it has no inhibitory effect on LTP.     

NMDA receptor antagonist-induced dopamine release in the ventral pallidum does not correlate with motor activation

The ventral pallidum is the output structure of the nucleus accumbens in the ventral corticostriato-thalamocortical loop. Information processing in this loop is critically involved in motor behavior and reinforcement. The ventral pallidum receives a direct dopaminergic input from the ventral tegmental area, but also glutamatergic input from cortical and limbic areas. It has been assumed that dopamine release in the VP is indeed modulated by glutamate. The present study investigated the effects of NMDA receptor blockade on motor behavior and dopamine release in the ventral pallidum. In a first experiment, rats were implanted with microdialysis probes in the ventral pallidum and were systemically injected or locally perfused via the microdialysis probe with dizocilpine (0.32 mg/kg, 10 and 100 microM, respectively). Effects on dopamine and on locomotion were simultaneously monitored. In a second experiment, ventral pallidum was lesioned by quinolinic acid and the effects of systemic dizocilpine (0.08 and 0.16 mg/kg) on locomotion and stereotyped sniffing behavior were determined. It was found that systemic and local dizocilpine administration increased dopamine release in the ventral pallidum to a similar extent whereas only systemic treatment was accompanied by locomotor stimulation. Lesion of the ventral pallidum did not affect locomotion and stereotyped sniffing behavior induced by systemic dizocilpine treatment. Thus, DA release in the ventral pallidum that is elevated by blockade of NMDA receptors is not relevant for activation of motor behavior.     

MK-801, but not drugs acting at strychnine-insensitive glycine receptors, attenuate methamphetamine nigrostriatal toxicity

Repeated administration of methamphetamine (METH) results in damage to nigrostriatal dopaminergic neurons. Both competitiveN-methyl-d-aspartate (NMDA) receptor antagonists and use-dependent cation channel blockers attenuate METH-induced damage. The objectives of the present study were to examine whether comparable reductions in METH-induced damage could be obtained by compounds acting at strychnine-insensitive glycine receptors on the NMDA receptor complex. Four injections of METH (5 mg/kg i.p.) resulted in a 70.9% depletion of striatal dopamine (DA) and 62.7% depletion of dihydroxyphenylacetic acid (DOPAC) content, respectively. A significant protection against METH-induced DA and DOPAC depletion was afforded by the use-dependent channel blocker, MK-801. The competitive glycine antagonist 7-chlorokynurenic acid (7-Cl-KA), the low efficacy glycine partial agonist (+)-3-amino-1-hydroxy-2-pyrrolidone ((+)-HA-966), and the high efficacy partial glycine agonist 1-aminocyclopropane-car☐ylic acid (ACPC) were ineffective against METH-induced toxicity despite their abilities to attenuate glutamate-induced neurotoxicity under both in vivo and in vitro conditions. These results indicate that glycinergic ligands do not possess the same broad neuroprotective spectrum as other classes of NMDA antagonists.