Abstract
Receptor binding studies and electrophysiological studies demonstrated the existence of at least two kappa opioid receptors, which have been designated kappa-1 andkappa-2. Several agonists and antagonists are selective for the kappa-1 receptor whereas no known ligands are selective for the kappa-2 receptor. In this study, thekappa opioid GR89,696 was tested in the guinea pig hippocampal slice preparation for kappa-1versuskappa-2 activity. The perforant path-evoked population spike in the dentate was use to evaluate activity at the kappa-1 receptor, and the Schaffer collateral-evoked N-methyl-d-aspartate (NMDA) receptor-mediated synaptic current in CA3 pyramidal cells was used to measure kappa-2 receptor activation. GR89,696 had no effect on the perforant path-evoked dentate population spike; however, it did reverse the effects of the selective kappa-1 agonist U69,593 when co-perfused over the slices. In the CA3, GR89,696 inhibited the NMDA receptor-mediated synaptic current. The inhibition was antagonized by naloxone. The EC50 for GR89,696 on the NMDA current was 41.7 nM (95% CL, 7.0–248 nM). These findings indicate that GR89,696 is an agonist for kappa-2 opioid receptors and an antagonist at kappa-1 receptors in the guinea pig hippocampus.
Previous work demonstrated that kappa-1 and kappa-2 opioid receptors were differentially distributed in the guinea pig hippocampus (Wagner et al., 1993). The kappa-1 opioid receptors reside primarily on the perforant path fibers which provide the main excitatory input to the dentate granule cells (Drake et al., 1994), but some kappa-1 receptor activity has been demonstrated on the mossy fibers (Castillo et al., 1996;Weisskopf et al., 1993). Activation of kappa-1 receptors results in inhibition of excitatory amino acid release from presynaptic terminals and a subsequent decrease in amplitude of excitatory postsynaptic potentials in dentate granule cells (Termanet al., 1994; Wagner et al., 1992, 1993) and CA3 pyramidal cells (Castillo et al., 1996; Weisskopf et al., 1993).
Kappa-2 opioid receptors, on the other hand, are primarily located in the CA3 region of the guinea pig hippocampus (Wagneret al., 1992). For the purposes of this study, we have defined kappa-2 receptors as the specific bremazocine binding that remains after mu, delta andkappa-1 receptors have been blocked. Specific binding is determined by displacement with the opioid antagonist naloxone. This definition is consistent with the definitions used by several other investigators (Nock et al., 1990, 1993; Rothman et al., 1992; Zukin et al., 1988). This operational description of kappa-2 opioid receptors is used because the receptor has not been cloned nor have selective ligands been developed for the receptor.
Activation of kappa-2 opioid receptors results in the inhibition of NMDA receptor-mediated synaptic currents in CA3 pyramidal cells (Caudle et al., 1994). Kappa-1 selective drugs such as U69,593 have no effect on the NMDA receptor-mediated current. These findings suggest that drugs selective forkappa-2 receptors may be useful for regulating NMDA receptor-mediated pathologies.
NMDA receptors are involved in learning and memory (Castillo et al., 1996; Weisskopf, et al., 1993) and in several neuropathologies. NMDA receptors mediate much of the damage that occurs to nervous tissue after stroke (Faden et al., 1989; Faden and Salzman, 1992; Rogawski, 1993), they are involved in epilepsy (Meldrum, 1994; Meldrum, 1993) and persistent pain (Ren and Dubner, 1993; Ren et al., 1992a, b), as well as several other conditions. Because there is the potential to regulate NMDA receptor function through kappa-2 opioid receptors and because there are no known selective kappa-2 opioid agonists, we have undertaken a search to find selective kappa-2 agonists.
Recently, GR89,696 was synthesized and demonstrated to be a very potent and selective kappa agonist in rabbit vas deferens (Nayloret al., 1993). GR89,696 was a potent neuroprotective agent in a mouse ischemia model (Birch et al., 1991), and we found that it was a potent antihyperalgesic agent in a rat foot inflammation model (Ho et al., 1997). In the inflammation study, GR89,696 potently inhibited the heat hyperalgesia associated with the inflamed paw, but did not influence the heat sensitivity of the noninflamed paw. This effect was also produced by bremazocine. In contrast, the selective kappa-1 opioid U69,593 had no effect on either paw. Mu and delta selective agonists inhibited the response to heat of both paws. Since GR89,696 and bremazocine were the only agents tested that were antihyperalgesic we concluded that they produced their effect through a kappa-2 subclass of opioid receptor (Ho et al., 1997).
The interesting pharmacological properties of GR89,696 evoked the question as to whether or not this agent was a selectivekappa-2 agonist. In this study, we attempted to answer this question by both binding and electrophysiological experiments in guinea pig hippocampal slices.
Materials and Methods
All experiments were approved by the Animal Care and Use Committee of the National Institute of Dental Research. These experiments complied with the National Institutes of Health guide for care and use of laboratory animals. Every effort was made to use the minimum number of animals possible.
Receptor binding.
Guinea pig brain membranes were prepared as described previously (Wagner et al., 1992). Whole guinea pig brains were homogenized in 10 ml/g of 50 mM tris (pH 7.4) and then centrifuged at 10,000 × g (4°C) for 30 min. The membranes were then resuspended in tris and stored at −70°C until needed. Each assay tube contained 0.5 mg membrane protein, 3 nM [3H]bremazocine or 3 nM [3H]U69,593, the appropriate blocking ligands and various concentrations of GR89,696 in 50 mM tris buffer (pH 7.4). The final volume of each assay tube was 1 ml. All assays were performed in triplicate and each experiment was repeated three times. In the experiments that required masking of opioid receptor subtypes (DAMGO, 1 μM; DPDPE, 1 μM; U69,593; 1 μM) were used to block mu,delta and kappa-1 opioid receptors respectively. Nonspecific binding was defined with either 10 μM naloxone or 10 μM GR89,696. Preliminary binding experiments demonstrated that both naloxone and GR89,696 displaced the same amount of [3H]bremazocine and [3H]U69,593 binding. Assay tubes were incubated for 90 min at room temperature, filtered through Whatman GF/B filters and the filters were then washed three times with 5 ml ice-cold tris buffer. The filters had been presoaked in 0.5% polyethlenimine. The filters were then counted in a scintillation counter. Binding constants (Ki ) were calculated from the equation:Ki = (EC50)/(1 + F/Kd ) with the statistical software PRISM (Graphpad Software inc., San Diego, CA). WhereF is the concentration of [3H]ligand and Kd is the dissociation constant of the ligand. We determined binding constants for the radioligands by saturation binding analysis. These experiments provided aKd for bremazocine of 0.7 nM for a single binding site and a Kd for U69,593 of 1.5 nM (data not shown). These binding data are consistent with results published previously (Nock et al., 1993).
Hippocampal slice preparation.
Male Hartley guinea pigs (150–300 g) were anesthetized with pentobarbital (50 mg/kg i.p.) and decapitated. The brains were rapidly removed and cooled under ice-cold Kreb’s bicarbonate buffer with the following composition (mM): NaCl, 124; KCl, 4.9; KH2PO4, 1.2; MgSO4, 2.4; CaCl2, 2.5; NaHCO3, 25.6; and glucose, 10. The buffer pH was equilibrated by bubbling with 95% O2/5% CO2. The brains were then sliced on a vibratome (500 μM) and the hippocampal slices were dissected from the remainder of the tissue and placed in a recording chamber. Kreb’s bicarbonate buffer was superfused over the slices at a rate of approximately 1 ml/min. The chamber was then warmed slowly to 34°C, and the slices were incubated for at least 1 h to allow residual pentobarbital to wash out of the slices (Caudle et al., 1994).
Whole-cell voltage clamp.
Patch recording electrodes were pulled to resistances of 2 to 10 megohm and filled with a recording solution of the following composition (mM): CsCl, 120; tetraethylammonium chloride, 20; CaCl2, 1; MgCl2, 2; EGTA, 10; HEPES, 10; ATP, 4; and GTP, 0.5. The pH of the recording solution was adjusted to 7.4 with CsOH. The cesium and tetraethylammonium chloride in the recording electrode were used to inhibit both voltage-activated and GABAb receptor-activated potassium currents. The patch electrodes were then lowered into the CA3 region of the hippocampal slice to form a seal on the cell bodies of pyramidal cells. After the formation of a seal to a cell body, the membrane was ruptured to obtain a whole-cell voltage clamp of the cells. To record NMDA receptor-mediated synaptic currents a concentric bipolar stimulating electrode (SNE 100, Rhodes Medical Instruments, Woodland Hills, CA) was placed in the stratum lacunosum moleculare (Schaffer collaterals) of the CA1 approximately 500 μ from the CA1/CA3 border. Stimuli consisted of single 0.3-ms square waves with currents ranging from 200 to 400 μA. The intensity of the stimulus was adjusted to the minimum current necessary to produce the maximum NMDA receptor-mediated current. To isolate the NMDA receptor-mediated currents the Kreb’s bicarbonate buffer was changed to Kreb’s bicarbonate buffer containing nominally zero magnesium, 10 μM CNQX and 20 μM Bicuculline. The magnesium concentration was lowered to improve the consistency of the NMDA receptor-mediated currents. Fluctuations in the potential of the dendrites in the voltage-clamped cells may alter the level of magnesium block in the NMDA receptor channels. Removal of magnesium eliminates this problem. Bicuculline and CNQX were used to block GABAa and non-NMDA excitatory amino acid receptors, respectively. Finally, to inactivate voltage-activated sodium and calcium channels the cells were depolarized to +5 mV for 6 to 20 s before and during recording. All data collected were averages of five events. The recorded sweeps were 960 ms. The data were collected by an Axopatch 200 (Axon Instruments, Foster City, CA), digitized and recorded on a personal computer for future analysis. Current data were converted into area over the curve for all analyses. Area over the curve was used rather than area under the curve because the currents were inward, which resulted in a downward deflection of the current trace. All drugs were applied via the superfusion buffer (Caudle et al., 1994). The drugs were applied for 10 min before the initiation of recordings. Concentration-response relationships were analyzed with the statistical software PRISM (Graphpad Software inc., San Diego, CA). The equation for curve fitting was Y = Min + (Max − Min)/(1 +1 0 (Log (EC50) − Log (X))), where Max is the maximum effect produced by the drug and Min is the response in the absence of drug. The EC50 was determined by an iterative process to best fit the data.
Extracellular recording of dentate population spikes.
Extracellular recording electrodes were pulled to resistances of 2 to 5 megohms and filled with 3 M NaCl. The recording pipette was placed in the granule cell layer of the dentate gyrus and the stimulating electrode was placed at the apex of the stratum lacunosum moleculare of the dentate. Population spikes were evoked by 0.3-ms square waves ranging in intensity from 25 to 300 μA. Stimulus-response relationships were performed before drug application to confirm that the relationship was biphasic (Wagner et al., 1993). If the stimulus-response relationship was not biphasic the slice was assumed to be unhealthy and was discarded. All pharmacological experiments in the dentate gyrus were performed with the stimulus that produced the maximum amplitude population spike. Previous work demonstrated that the peak of the biphasic stimulus-response curve provided the most sensitive assay for kappa-1 opioid agonists (Wagner et al., 1992). All the data were collected by an Axopatch 200 (Axon Instruments, Foster City, CA) in current clamp mode. The data were digitized and stored on a personal computer for future analysis. All collected data represented averages of five events. All drugs were applied via the superfusion buffer. The drugs were applied for 10 min before the initiation of recordings. Population spike amplitudes were determined by first drawing a line between the two positively directed peaks. A vertical line was then drawn from the point on the first line directly above the peak of the population spike to the bottom of the potential. The length of the vertical line was used as the amplitude of the potential.
Drugs.
DAMGO, DPDPE, 2-amino-5-phosphonovalerate, Bicuculline and naloxone were purchased from Sigma Chemical Company (St. Louis, MO). Bremazocine, nor-binaltorphimine and U69,593 were purchased from Research Biochemicals Inc. (Natick, MA). CNQX was purchased from Tocris Cookson Inc. (St. Louis, MO). GR89,696 was a generous gift from Dr. B.M. Bain, Glaxo Research and Development Limited (Middlesex, UK). [3H]Bremazocine and [3H]U69,593 were purchased from New England Nuclear (Boston, MA).
Results
Displacement of [3H]bremazocine and [3H]U69,593 binding by GR89,696.
In whole guinea pig brain membranes GR89,696 displaced all the specific opioid binding of [3H]bremazocine (fig.1A). Specific binding was defined as the [3H]bremazocine that could be displaced by 10 μM naloxone or 10 μM GR89,696. The data were best fitted with a two-site binding model. The Ki for the high-affinity site was 2.3 nM (95% CL, 0.89–6.0 nM) and theKi for the low-affinity site was 55.9 nM (95% CL, 19.7–158 nM). The high-affinity site, presumably bothkappa opioid receptors, represented 49.5 ± 10.8% of specific bremazocine binding. To examine the displacement of [3H]bremazocine from kappa-2 receptors the mu, delta and kappa-1 receptors were blocked by DAMGO (1 μM), DPDPE (1 μM) and U69,593 (1 μM), respectively (fig. 1B). Kappa-2 receptor binding represented 14.7 ± 1.2% of total specific bremazocine binding in guinea pig brain membranes. The calculatedKi for GR89,696 at the kappa-2 receptor was 6.3 nM (95% CL, 2.0–19.1 nM) (fig. 1B). To examine GR89,696 displacement of [3H]bremazocine fromkappa-1 receptors the mu and deltareceptors were blocked with 1 μM DAMGO and 1 μM DPDPE. The displacement curve for kappa-2 receptors was then subtracted from the resulting displacement curve to provide the curve displayed in figure 1C for kappa-1 receptors. The kappa-1 receptors represented 18.7 ± 2.0% of the total specific bremazocine binding, and the calculated Ki for GR89,696 was 0.5 nM (95% CL, 0.17–1.5 nM). The kappa-1 binding data were confirmed by the displacement of [3H]U69,593 by GR89,696 (fig. 1D). The calculated Ki for GR89,696 in displacing [3H]U69,593 was 0.4 nM (95% CL, 0.24–0.57 nM). The displacement curve of [3H]bremazocine from mu and delta receptors by GR89,696 was determined in the presence of 1 μM U69,593. The kappa-2 curve was then subtracted from the resulting curve to yield the curve displayed in figure 1C. Mu and delta receptors represented 58.8 ± 1.4% of total specific bremazocine binding and the calculated Ki was 37.1 nM (95% CL, 24.0–57.4 nM).
Effect of GR89,696 on NMDA receptor-mediated synaptic currents in CA3 pyramidal cells.
The selectivity of the NMDA receptor-mediated current as an assay for kappa-2 opioid receptors was confirmed by applying various selective opioid agonists to the superfusion media. As illustrated in figure2, the kappa-1 selective agonist U69,593 (1 μM, n = 3) had no effect on the NMDA receptor-mediated current, whereas the delta selective agonist DPDPE (1 μM, n = 3) and the muselective agonist DAMGO (1 μM, n = 3) increased the current. The nonselective kappa opioid agonist bremazocine (1 μM, n = 3) was very effective at inhibiting the current. These findings demonstrate that activation ofkappa-2 opioid receptors inhibits the NMDA receptor-mediated synaptic current. The data are consistent with the previous characterization of the kappa-2 opioid receptors in this preparation (Caudle et al., 1994).
When the novel kappa agonist GR89,696 was applied to the slice via the superfusion buffer the NMDA receptor-mediated current was strongly inhibited (see sweep in fig.3). The inhibition was concentration dependent (analysis of variance, F = 8.7, dF = 14, P = .0028) with an EC50 of 41.7 nM (95% CL, 7.0–248 nM) (fig. 3). The effects of GR89,696 on thekappa-2 opioid receptors were antagonized by 1 μM naloxone (n = 3), but not by the selective kappaantagonist nor-binaltorphimine (1 μM, n = 3) (fig.4).
Effect of GR89,696 on the dentate population spike.
To determine whether GR89,696 was an agonist at the kappa-1 opioid receptor, perforant path-evoked population spikes were recorded from the guinea pig dentate gyrus (Terman et al., 1994;Wagner et al., 1992,1993). In this assay, the selectivekappa-1 agonist U69,593 (1 μM, n = 3) and the nonselective kappa agonist bremazocine (1 μM,n = 4) inhibited the population spike (fig.5). The effects of U69,593 and bremazocine were reversed by 1 μM nor-binaltorphimine. GR89,696 (10 μM, n = 5), on the other hand, was ineffective at reducing the amplitude of the population spike. These data suggest that GR89,696 is not an agonist at the kappa-1 opioid receptor in the guinea pig hippocampus. Because GR89,696 clearly binds tokappa-1 opioid receptors (fig. 1), it was hypothesized that GR89,696 may be an antagonist or a partial agonist at thekappa-1 opioid receptor. To test this hypothesis, 10 μM U69,593 was added to the bathing solution. After the U69,593 inhibited the dentate population spike 10 μM GR89,696 was added to the solution in an attempt to antagonize the effects of U69,593. U69,593 reduced the amplitude of the dentate population spike to 53 ± 17% (n = 3) of control (P < .05, paired ttest). The addition of GR89,696 to the bathing solution reversed the inhibition to 89 ± 13% of control (P > .05, pairedt test when compared with control) (fig.6).
Discussion
In this study, we found that GR89,696 is an agonist forkappa-2 opioid receptors and an antagonist atkappa-1 opioid receptors in the guinea pig hippocampus. GR89,696 inhibited the NMDA receptor-mediated current in CA3 pyramidal cells. As described previously (Caudle et al., 1994) and demonstrated in figure 2, the only opioid agonists that inhibit the NMDA receptor-mediated current in the CA3 pyramidal cells are agents that act at the kappa-2 receptor. The kappa-1 selective agonist U69,593 has no effect on the NMDA receptor-mediated synaptic current. The selectivity of U69,593 for kappa-1 receptors versus kappa-2 receptors is greater than 10,000-fold (Nock et al., 1993). Thus, the lack of effect of U69,593 indicates that kappa-1 receptors are not involved in inhibition by GR89,696 of the NMDA receptor-mediated current. In addition, the effects of GR89,696 on NMDA receptor-mediated currents could only be blocked by the nonselective opioid antagonist, naloxone. The selective kappa-1 antagonist nor-binaltorphimine did not significantly block the effects of GR89,696. The selectivity of nor-binaltorphimine for kappa-1versus kappa-2 receptors is approximately 200-fold (Nock et al., 1993). Mu anddelta selective agonists enhance the NMDA receptor-mediated current. Hence, the conclusion that GR89,696 is a kappa-2 agonist was determined by a process of elimination. The data presented here for GR89,696 are consistent with previous work characterizing thekappa-2 opioid receptor in the CA3 region of the guinea pig hippocampus (Caudle et al., 1994). Therefore, GR89,696 is an agonist at the kappa-2 opioid receptor.
In the receptor binding experiments, GR89,696 was found to displace all specific bremazocine binding. The displacement data in the absence of any blocking agents (fig. 1A) was best fitted by a two binding site model. Presumably, the high-affinity site represents both thekappa-1 and kappa-2 receptors, and the low-affinity site represents the mu and deltareceptors. This interpretation is confirmed by the experiments with selective blocking agents. The percentage of bremazocine binding tokappa sites in the experiments with blocking agents was similar to the predicted high-affinity sites in the experiments with no blocking agents (33.4% and 49.5%, respectively). TheKi for GR89,696 at the high-affinity site in the experiments without blocking agents was 2.3 nM. If we assume there was a single kappa receptor subtype, theKi for GR89,696, calculated from the binding data obtained when mu and delta receptors were blocked, was 1.6 nM. If two kappa subclasses are assumed, the Ki values are 0.5 and 6.3 nM. These calculations confirm that the high-affinity binding site from the [3H]bremazocine experiments without blocking agents represents both the kappa-1 and kappa-2 receptors. In addition, GR89,696 had a Ki of 0.4 nM when displacing [3H]U69,593 fromkappa-1 receptors. This finding is consistent with theKi for GR89,696 at kappa-1 receptors when [3H]bremazocine was used as the radioligand (0.5 nM). Thus, the data are consistent with two subtypes of kappa opioid receptor. The mu anddelta receptors represented 58.8% of specific bremazocine binding in the experiments with blocking agents and 50.5% in the experiments without blocking agents. TheKi values for the two sets of data were 37.1 nM and 55.9 nM, respectively. The good agreement between the two types of binding experiments confirms that the blocking agents were effective at blocking their respective receptors and that GR89,696 has high affinity for both kappa opioid receptors.
In light of the high-affinity of GR89,696 for kappa-1 receptors it was surprising to find that GR89,696 was ineffective at inhibiting the dentate population spike. The dentate population spike was previously demonstrated to be a good assay for kappa-1 opioid receptor agonist activity (Drake et al., 1994; Termanet al., 1994; Wagner et al., 1992, 1993). And, U69,593 and bremazocine effectively inhibited the population spike in a nor-binaltorphimine-reversible manner (see fig. 5). Thus, the finding that GR89,696 did not inhibit the dentate population spike suggested that GR89,696 was an antagonist, or partial agonist, at thekappa-1 site. This hypothesis was confirmed by GR89,696 antagonizing the inhibition by U69,593 of the dentate population spike. No inhibition of the dentate population spike was observed with concentrations of GR89,696 ranging from 1 nM to 100 μM (n = 20 hippocampal slices, unpublished observations). Because 100 μM GR89,696 should saturate kappa-1 opioid receptors it is reasonable to assume that this agent is akappa-1 antagonist in the guinea pig dentate gyrus. Alternatively, activation of kappa-2 receptors in the dentate may physiologically antagonize kappa-1 receptor inhibition of the population spike. However, bremazocine has substantial agonist activity at kappa-2 receptors, yet it effectively inhibits the dentate population spike. Therefore, it would be unlikely that the kappa-1 receptor antagonism of GR89,696 is produced indirectly through kappa-2 receptors.
There are several potential uses for kappa-2 agonists that would be of clinical significance. Mu and deltaagonists enhance NMDA receptor-mediated currents (Caudle et al., 1994; Chen and Huang, 1994), and NMDA antagonists can block tolerance to morphine, reduce the withdrawal symptoms of morphine and block epileptiform activity produced by normorphine (Bhargava and Matwyshyn, 1993; Swearengen and Chavkin, 1987; Trujillo and Akil, 1991,1994; Yukhananov and Larson, 1994). Thus, the use of conventional opioids to treat conditions that have an underlying NMDA receptor-mediated component results in a counter-productive enhancement of the NMDA component. It is well documented that NMDA receptors are involved in chronic pain processes (Ren and Dubner, 1993; Renet al., 1992a, b), epilepsy (Meldrum, 1993, 1994; Rogawski, 1993) and traumatic injury to the central nervous system (Fadenet al., 1989; Faden and Salzman, 1992). Therefore, a selective kappa-2 agonist should be useful in treating these conditions and should be devoid of the side effects and counter-productive NMDA-enhancing properties of conventional opioids. Recently, we demonstrated that GR89,696 is a potent antihyperalgesic agent when injected intrathecally into rats (Ho et al., 1997). In rats with inflammation in one hind paw, intrathecal administration of GR89,696 reversed the hyperalgesia to heat in the inflamed paw, but did not influence the response to heat of the noninflamed paw. Selective mu and delta agonists inhibited the response to heat of both hind paws whereas selectivekappa-1 agonists had no effect. These results are similar to those observed with NMDA receptor antagonists (Ren and Dubner, 1993;Ren et al., 1992a, b), which suggests that thekappa-2 receptors may inhibit NMDA receptors in the spinal cord as they do in the CA3 region of the guinea pig hippocampus. GR89,696 has also proven itself to be effective in reducing the damage associated with cerebral ischemia (Birch et al., 1991). This observation may also be a result of reduced current flow through NMDA receptors when GR89,696 activates kappa-2 receptors.
In summary, although GR89,696 is not a selective ligand for thekappa-2 opioid receptor, its unique spectrum of antagonism at kappa-1 and agonist activity at kappa-2 opioid receptors makes it a useful tool for examining the function ofkappa-2 receptors. From the studies that have used GR89,696 it is clear that kappa-2 receptors may represent an important route to the regulation of NMDA receptor function in certain neuropathologies.
Acknowledgments
We thank Dr. B.M. Bain (Glaxo Research and Development Limited) for the gift of GR89,696 and Drs. Diana Douglass and Ke Ren for their advice in the preparation of this manuscript.
Footnotes
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Send reprint requests to: Robert M. Caudle, PNMB/NIDR/NIH, Building 49, Room 1W26, 49 Convent Drive, MSC 4410, Bethesda, MD 20892-4410.
- Abbreviations:
- CNQX
- 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione
- DAMGO
- [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin
- DPDPE
- [d-Pen2,5]enkephalin
- NMDA
- N-methyl-d-aspartate
- U69
- 593, N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide
- GR89
- 696, methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl)methyl]-1-piperazinecarboxylate
- EGTA
- ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- GABA
- γ-aminobutyric acid
- CL
- confidence limit
- Received April 18, 1997.
- Accepted August 28, 1997.
- The American Society for Pharmacology and Experimental Therapeutics