Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Glutamate is the dominant excitatory neurotransmitter of the mammalian central nervous system. Almost all types of central neurons can be excited by glutamate acting on a variety of ligand-gated ion channels or G protein-coupled (metabotropic) receptors (Hollmann and Heinemann, 1994). Glutamatergic transmission is critically involved in many important events of the central nervous system, such as synaptic plasticity in the developing and adult brain, as well as neuronal survival and death (Choi, 1994; Collingridge and Bliss, 1995). The glutamate-gated ion channel receptors have been classified into three main categories, those sensitive to the agonist N-methyl-D-aspartate (NMDA; NMDA receptors), those sensitive to the agonist kainate (kainate receptors), and those sensitive to the agonist -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; AMPA receptors). Molecular cloning studies have revealed that NMDA receptors are heteromultimeric protein complexes, composed of at least two subunits, NMDAR1 (NR1) and NMDAR2 (NR2). Whereas NR1 exists in several variants generated by alternative splicing from a single gene, four distinct genes are responsible for the expression of NR2A, 2B, 2C, and 2D (Hollmann and Heinemann, 1994). The NMDA receptor is blocked by external physiological Mg²⁺ at resting membrane potential. Depolarization removes this block, allowing entry of Ca²⁺, which can in turn activate a variety of signal transduction pathways (Mayer et al., 1984). As a consequence of this mechanism, NMDA receptors can function as detectors of temporally coincident synaptic inputs to the same neuron. Such a cascade of events is thought to be at the basis of the long-term potentiation of synaptic inputs to CA1 pyramidal neurons in the hippocampus, an event responsible for certain forms of learning (Herron et al., 1986). However, the crucial role of NMDA-based detectors of coincident activity is not restricted to hippocampal synaptic plasticity. Indeed, evidence is present to support the crucial role of the NMDA receptors in other forms of neuronal plasticity induced by prolonged electrical activity. Peripheral tissue injury or inflammation induces a state of sensory hypersensitivity that manifests itself as allodynia (decreased pain threshold) and hyperalgesia (an increased response to noxious stimuli). This pain hypersensitivity results from both an increase in transduction sensitivity of primary afferent receptors and an increase in the excitability of spinal cord neurons (Ma and Woolf, 1995). Several lines of investigation have shown the involvement of the glutamatergic system in the development and maintenance of pain hypersensitivity. Iontophoretic applications of glutamate produce a facilitation of responses to low and high intensity of mechanical stimulation of the skin (Dougherty and Willis, 1991), whereas an increase in glutamate release has been observed in rat spinal cord after injection of an irritant agent or nerve injury (Sluka and Westlund, 1992; Malmberg and Yaksh, 1995). Moreover, it appears that the dorsal horn neuronal plasticity and hyperexcitability after tissue injury involve the effect of excitatory amino acid on NMDA receptors. In fact, NMDA antagonists have been shown to suppress formalin-induced pain behavior (Haley et al., 1990), hyperalgesia induced by chronic constriction injury (CCI) to the rat sciatic nerve (Davar et al., 1991), and peripheral inflammation (Ren et al., 1992).

The NMDA receptor is unique because opening of the channel requires the simultaneous binding of glutamate and glycine (Corsi et al., 1996). Therefore, the NMDA receptor blockade can also be obtained through the antagonism of the glycine site, which may represent a centerpiece for a novel strategy to explore in vivo the role of NMDA receptors (Dickenson and Aydar, 1991; Ren et al., 1992). Therefore, to further study the role of NMDA receptors in chronic pain, we have used a novel, potent, and selective antagonist of the glycine site, GV196771A, [E-4,6-dichloro-3-(2-oxo-1-phenyl-pyrrolidin-3-ylidenemethyl)-1H-indole-2-carboxylic acid sodium salt, Fig. 1; Giacobbe et al., 1998].

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structure of GV196771A.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Binding Studies

Animals and Tissue Preparation. Male Sprague-Dawley rats (200-250 g) were used. Animals were supplied by Charles River (Lecco, Italy) and were allowed food and water until used. Immediately after sacrifice, brains were removed and used for the preparation of cerebral cortex synaptic membranes to be used in radioligand binding studies.

[³H]Glycine Binding Assay. [³H]Glycine displacement experiments were performed as described by Mugnaini et al., (1998). GV196771A was dissolved in dimethyl sulfoxide (DMSO) at 5 mM and tested at seven concentrations in duplicate (0.1 nM to 100 µM) in five separate experiments.

Antagonism of Glycine-Induced [³H]TCP Binding Enhancement. [³H]TCP binding experiments were performed as follows. To minimize glycine contamination all steps were performed in laboratory glassware previously treated at high temperature (250°C) for more than 4 h or treated with 1 M HCl and then extensively washed with fresh MilliQ water. Briefly, on the day of the experiment, rat cerebral cortex membranes were homogenized in 20 volumes of 5 mM Tris/HCl (pH 7.7) and centrifuged at 48,000g for 15 min. Pellets were then given 4 cycles of washing, each cycle consisting of resuspending the membranes in 20 volumes of buffer, incubating at 25°C for 20 min, and centrifuging at 48,000g for 15 min. Glycine concentration response curves (CRC) at enhancing [³H]TCP binding have been obtained in the presence of increasing concentration of GV196771A and 1 µM glutamic acid. Final pellets were resuspended in 30 volumes of buffer and [³H]TCP binding was performed in a final volume of 1000 µl containing: 180 µl of buffer, 100 µl of buffer with 1 µM glutamic acid (final concentration), 20 µl of buffer containing increasing concentrations of glycine (from 0.1 nM to 100 µM final concentration, including intermediate concentrations), 100 µl of buffer with 10X the final desired concentration of GV196771A, 100 µl of buffer with the radioligand ([³H]TCP at a final concentration of 2.5 nM), and 500 µl of final membrane suspension. The reaction was allowed to proceed for 2 h at 30°C and then stopped by filtration through glass fiber filters (GF/C, Whatman International, Maidstone, UK) on a Brandel M48R cell harvester (Brandel, Gaithersburg, MD), followed by rapid washing of the filters two times with 3 ml of ice-cold buffer. Filters were collected in polyethylene vials (Biovials; Beckman Instruments, Berkeley, CA) to which 3.5 ml of scintillation fluid (Filter Count, Packard Instruments, Downers Grove, IL) were added. The vials were capped, shaken, and counted in a Packard TRI-CARB 1900 CA liquid scintillation analyzer. CRC to glycine in the absence (control) or presence of antagonist (0.3, 1, and 3 µM GV196771A) were simultaneously obtained on the same experiment with each incubation performed in triplicate.

[³H]AMPA, [³H]-cis-4-(Phosphonomethyl)-2-piperidinecarboxylic acid (CGS-19755), and [³H]Kainic Acid Binding Assay. [³H]AMPA and [³H]-CGS-19755 displacement experiments were performed essentially as described previously by Giberti et al. (1991) and Murphy et al. (1988). [³H]Kainic acid displacement experiments were performed as follows. Membranes for the [³H]kainic acid binding assay were incubated (60 min, 4°C) in Tris/acetate 50 mM (pH 7.10) with 2 nM radioligand. The reaction was stopped by dilution with ice-cold buffer solution and filtration as indicated for [³H]TCP binding assay.

Data Analysis for Binding Experiments. Data of [³H]glycine and [³H]kainic acid displacement experiments were analyzed using the nonlinear curve fitting program Ligand (Munson and Rodbard, 1980) to determine the inhibition constants of displacer ligands (K_i); the required K_D values of the radioligands were set to 177 nM and 2.19 nM, as determined previously in saturation studies with [³H]glycine and [³H]kainic acid, respectively (data not shown). Given the ability of [³H]AMPA to label two populations of binding sites in the conditions used in this study (Giberti et al., 1991) and the consequent complexity of estimation of K_i values, data of [³H]AMPA displacement experiments were analyzed using Allfit (De Lean et al., 1978) and only the concentration of displacer ligands inhibiting 50% of [³H]AMPA-specific binding (IC₅₀) were estimated. K_i and IC₅₀ values have been expressed as pK_i (LogK_i) and pIC₅₀ (Log IC₅₀) ± S.E.M. In [³H]TCP binding experiments, CRCs to glycine in the presence of increasing concentrations of GV196771A were simultaneously fitted to the following equation:

(1)

where [A] is the concentration of agonist, R_max is the maximal glycine-induced [³H]TCP binding, EC₅₀ is the agonist concentration capable of producing 50% of maximal response, n is the Hill slope factor of the glycine CRCs, [B] is the concentration of antagonist, m is the "Schild plot" slope parameter (Arunlakshana and Schild, 1959), and K is a parameter which measures the potency of the antagonist and corresponds to the apparent equilibrium dissociation constant of the antagonist (K_B) when m = 1. Curves were first fitted to the above general model, allowing the parameter m to be estimated. Subsequently, if the estimated m parameter was not significantly different from unity, data were re-fitted with the model where m was constrained to unity, and a K_B value could be estimated. The K_B value has been expressed as pK_B (LogK_B) followed by 95% C.L. Calculations were performed with the data manipulation and analysis software RS1 (Bolt, Beranek and Newman, Boston, MA).

Drugs and Solutions for Binding Experiments. [³H]Glycine (NET 004, specific radioactivity 1620.6 GBq/mmol), [³H]AMPA (NET 833, specific radioactivity 1961.0 GBq/mmol), [³H]kainic acid (NET 875, specific radioactivity 2146.0 GBq/mmol), [³H]TCP (NET 886, specific radioactivity 1835.2 GBq/mmol), and [³H]-CGS-19755 (NET 988, specific radioactivity 9.25 MBq/nmol) were obtained from DuPont-NEN (Boston, MA). GV196771A was synthesized by the Medicinal Chemistry department of Glaxo Wellcome S.p.A. (Verona, Italy). Glycine, glutamate, and various salts were purchased from Sigma Chemical Company (St. Louis, MO). All salts and reagents were of the highest analytical grade available.

Electrophysiological Recordings

We have used the whole cell patch-clamp recording technique to measure macroscopic currents. The preparations used included primary cultures of rat embryonic neurons. Moreover, in parallel experiments, we have used extracellular recordings from the ventral root of isolated baby rat spinal cords to measure spinal cord wind-up.

Culture of Cortical Neurons. Neurons were prepared as described by Tang and Aizenman (1993). Briefly, cortices of 16-day-old Sprague-Dawley embryonic rats (Charles River) were incubated in a dissociation medium [minimal essential medium (MEM) containing 2 mM glutamine, 1% penicillin/streptomycin, and 0.2% glucose] containing 0.032 mg/ml trypsin for 2 h. The tissue was then incubated for 20 min in Earle's balanced salt solution and dissociated by means of a Pasteur pipette. Cells were suspended in plating medium (Dulbecco's medium, supplemented with 10% calf serum, 10% nutrient mixture F-12 ham medium, 2 mM glutamine, 1% penicillin/streptomycin, and 25 mM HEPES) and plated onto glass coverslips coated with 10 mg/ml poly-l-lysine. The culture was treated on day 15 with 1.5 µg/ml cytosine-B-D-arabinofuranoside. Half changes of medium were done three times a week.

Culture of Hippocampal Neurons. Cells were prepared as described by Goslin and Banker (1991). Briefly, hippocampi of 18-day-old Sprague-Dawley embryonic rats (Charles River) were incubated in a low-calcium saline with trypsin for 20 min. The trypsin was inactivated with MEM supplemented with 10% horse serum, 2 mM glutamine, 1% penicillin/streptomycin, and 0.6% glucose (plating medium). The tissue was mechanically dissociated and plated onto a confluent layer of cortical glial cells. The second day in culture the plating medium was substituted with feeding medium (MEM containing 2 mM glutamine, 1% penicillin/streptomycin, 0.6% glucose, 1 mg/ml bovine serum albumin, 0.1 mg/ml transferrin, 100 µM putrescine, 30 nM Na-selenite, 0.5 mg/ml insulin, 20 nM progesterone, and 1 mM Na-piruvate).

Culture of Spinal Cord Neurons. Spinal cord neurons were prepared as described by Fitzgerald (1989). Briefly, spinal cords of 14-day-old Sprague-Dawley embryonic rats (Charles River) were incubated in a low-calcium saline with trypsin for 30 min. The enzyme was inactivated in a plating medium consisting of MEM supplemented with 10% fetal bovine serum, 10% horse serum, 0.37% Na-bicarbonate, 0.6% glucose, 2 mM glutamine, and 40 µg/ml deoxyribonuclease I. The tissue was mechanically dissociated and plated onto 10 mg/ml poly-l-lysine-coated glass coverslips. The next day the plating medium was substituted with a feeding medium composed of MEM with 0.37% Na-bicarbonate, 0.6% glucose, 2 mM glutamine, 5% horse serum, 10 µg/ml bovine serum albumin, 0.2 mg/ml transferrin, 32 µg/ml putrescine, 10 ng/ml Na-selenite, 20 ng/ml triiodothyronine, 10 ng/ml insulin, 12 ng/ml progesterone, and 40 ng/ml corticosterone. All cultures were treated 5 days after plating with a mixture of 5'-fluoro-2-deoxyuridine and uridine (20 and 50 µg/ml, respectively) to suppress overgrowth of background cells. Half changes of medium were done twice weekly.

Isolation of Rat Neonatal Spinal Cords. Spinal cords were prepared from 4- to 10-day-old Sprague-Dawley rats (Charles River) as described by Thompson et al. (1992).

Electrical Recordings. Whole cell patch-clamp recordings were performed on cultured neurons after 1 week. The extracellular solution contained (in mM): NaCl 140, KCl 5, CaCl₂ 1, HEPES 10, glucose 10, pH adjusted to 7.4. Tetradotoxin (0.1 µM) was used to block spontaneous activity. The intracellular (pipette) solution contained (in mM): CsCl 140, EGTA 11, MgCl₂ 4, Mg-ATP 2, HEPES 10, pH adjusted to 7.3. The recording chamber was placed on the stage of an inverted microscope and continuously superfused by gravity with the extracellular solution containing glycine and GV196771A at the specified concentrations. Test NMDA solution was applied rapidly with the "U-tube" method, positioning the ejection hole within 100 to 200 µm of the cell. The cells were voltage-clamped at 60 mV. Only cells having a resting potential more negative than 40 mV were used. Currents were digitized (100 Hz), low-pass filtered (40 Hz), and stored on-line using an IBM-compatible PC running pClamp (Axon Instruments, Foster City, CA).

Data Analysis for Electrophysiology. Current amplitude was measured and analyzed by means of custom made analysis software written in Axobasic (Axon Instruments). All current measurements, unless otherwise stated, refer to the steady-state value of the responses (Iss) and are the average of the last 3 to 4 s of agonist application. For the generation of the agonist CRC, values were expressed as a percentage and normalized to the maximal effect. Glycine CRCs were obtained in the presence of 100 µM NMDA, whereas NMDA CRCs were obtained in the presence of 3 or 10 µM glycine (as indicated). Data were fitted to the equation to estimate the response at the steady state:

(2)

where I_min is the response obtained in the absence of added agonist, I_max is the maximal response, [A] is the agonist concentration, [EC₅₀] is the agonist concentration that gives 50% of the maximal response and n is the Hill slope factor.

(3)

(4)

Drug and Solution for Electrophysiological Studies. NMDA, morphine hydrochloride, and glycine were purchased from Sigma, whereas L-AP5 was purchased from Tocris (Langford, UK). GV196771A was stored at 10 mM either in a solution of 50% DMSO-50% distilled water (patch-clamp experiments) or 100% DMSO (wind-up experiments). The maximal concentration of DMSO present in the final solutions was 0.5% for patch-clamp experiments and 0.1% for wind-up experiments. We found that at these concentrations DMSO did not affect the measured responses.

Behavioral Studies

Effect of GV196771A on Thermal Hyperalgesia in A Rat Model of Painful Mononeuropathy: CCI Model. Male Sprague-Dawley rats (Charles River) weighing 200 to 300 g were used. Animals were housed in groups of 2 to 3 and fed with chow pellet diet with free access to water. They were fasted overnight before the study and were allowed free access to water. Rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.). The left common sciatic nerve was exposed, and proximal to the sciatic trifurcation about 10 mm of nerve was freed of adhering tissue and four ligatures (3.0 chromic gut) were tied loosely around it with about 1 mm of spacing (Bennett and Xie, 1988). Rats were tested for thermal hyperalgesia using a commercial available analgesimeter (Plantar test, Ugo Basile, Comerio, Italy) by applying a heat stimulus (50W, 8V) directed onto the plantar surface of each hind paw, and the paw withdrawal latency (s) was determined. Four latency measurements were taken for each hind paw and averaged. The results were expressed as the difference score (DS) by subtracting the latency of the control side from the latency of the ligated side. Negative DSs indicated a lower threshold on the ligated side supporting an hyperalgesic state. The animals developed thermal hyperalgesia within 14 to 21 days after surgery.

Effect of GV196771A and Morphine on Pain Behavior in Mice Paw Formalin Test: Acute Administration. Male albino CD mice (Charles River) weighing 25 to 30 g were used. Animals were housed in groups of 5 to 6 and fed with chow pellet diet with free access to water. They were fasted overnight before the study and were allowed free access to water.

Effect of Chronic Treatment with GV196771A or Morphine in Mice Paw Formalin Test. Mice were divided randomly into seven groups (10-21 mice per group) and administered once daily for 8 days as follows: groups g1 and g3 received saline i.p.; groups g2 and g5 received methocel p.o.; group g4 received morphine 10 mg/kg i.p.; and groups g6 and g7 received GV196771A 3 and 10 mg/kg p.o., respectively (Table 1). On day 9 mice were treated with saline i.p. (g1), methocel po (g2), morphine 3 mg/kg i.p. (g3 and g4), and GV196771A 3 mg/kg (g5, g6, and g7); the protocol treatment is described in Table 1. GV196771A 3 mg/kg p.o. was administered 1 h before formalin injection whereas morphine 3 mg/kg i.p. was administered 30 min before formalin injection. The evaluation of the drugs effects was carried out by a blind operator.

Statistic Analysis for Behavioral Studies. For all experiments the data are expressed as mean ± S.E.M.

CCI test: prophylactic treatment. Statistical analysis was performed within each group to compare DS calculated at each time point after ligation versus basal values before surgery. In addition, the time course of DS of the vehicle-treated group was compared with the GV196771A-treated group. One-way ANOVA followed by Dunnett's test, where p < .05 was considered significant, was used.

CCI test: therapeutic treatment. Statistical analysis was performed to compare DS of control response (vehicle) and GV196771A-treated groups, taken at various time after administration, versus pretreatment values, using one-way ANOVA followed by Dunnett's test where p < .05 was considered significant. Dose-response curve regression analysis was then performed to evaluate regression line parameters to calculate the ED₅₀ (dose of GV196771A in mg/kg that reduced the thermal hyperalgesia by 50%) and 95% confidence intervals.

Formalin test: acute treatment. Statistical analysis was performed to compare control response (vehicle) with test response using one-way ANOVA followed by Dunnett's test where p < .05 was considered significant. Dose-response curve regression analysis was then performed to evaluate regression line parameters to calculate the ED₅₀ (dose of GV196771A or morphine, in mg/kg, that reduced the licking time by 50%) and 95% confidence intervals.

Formalin test: chronic treatment. Statistical analysis was performed to evaluate the following comparisons: 1) g1 with g3 and g4; 2) g2 with g5, g6, and g7; and 3) g5, g6, and g7 against each other.

Drugs and Solutions for Behavioral Studies. GV196771A was prepared as a stock solution of 1 mg/ml in 0.5% methylcellulose (methocel); further dilutions were prepared in 0.5% methocel. Morphine (Sigma) was prepared as a stock solution of 10 mg/ml in saline; further dilutions were prepared in saline.

	Results

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Binding Experiments

[³H]Glycine. To characterize GV196771A as a glycine site antagonist, we studied its effects on the binding of glycine in rat cerebral cortex membranes. It was observed that GV196771A inhibited [³H]glycine binding in a concentration-dependent fashion. Moreover, GV196771A was able to completely suppress the specific binding of glycine (Fig. 2A). The resulting pKi was 7.56 ± 0.09 (n = 5).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   A, displacement binding curve of [3H]glycine by GV196771A in rat cortical membranes. B, antagonism of GV196771A against glycine-induced enhancement of [3H]TCP binding. Control (), GV196771A 0.3µM (), GV196771A 1 µM (), and GV196771A 3 µM (). In [3H]TCP binding experiments a concentration of 1 µM glutamic acid was used.

[³H]TCP. Because TCP binds only to open NMDA receptors, labeled TCP can be used to measure the fraction of open channels independently from electrophysiological methods. In agreement with its expected behavior, very low specific [³H]TCP binding to rat cerebral cortex membranes was observed in the absence of glycine and glutamate. After addition of glutamic acid (1 µM) increasing concentrations of glycine (0.1 nM to 100 µM) progressively enhanced [³H]TCP binding until it reached a maximum. The glycine CRC had an estimated pEC₅₀ of 7.32 (7.27-7.39; 95% C.L.). The action of GV196771A was examined on TCP binding. In the presence of increasing concentrations of GV196771A (0.3-3 µM), parallel rightward shifts of the glycine CRC could be observed (Fig. 2B) with no significant depression of the maximal response. The Shild slope factor was not significantly different from unity (m = 1.09; 0.99-1.19; 95% C.L.) and a pK_B value of 7.13 (7.06-7.21; 95% C.L.) was estimated.

[³H]AMPA, [³H]-CGS-19755, and [³H]Kainic Acid. GV196771A did not displace [³H]AMPA and [³H]-CGS-19755 binding up to a concentration of 10 µM. Therefore, a pIC₅₀ <4 could be inferred for GV196771A for both AMPA and NMDA binding sites. In the same experiments, glutamic acid dose-dependently inhibited [³H]AMPA binding with an estimated pIC₅₀ of 6.64, and [³H]-CGS-19755 binding with an estimated pIC₅₀ of 7.90.

Electrophysiological Recordings

GV196771A Antagonizes NMDA/Glycine-Induced Currents in Embryonic Rat Neurons. Because the NMDA receptor regulates the gating of an intrinsic ion channel, one can quantitatively study the effect of GV196771A by measuring the currents flowing through this channel. In the primary cultures of cortical, hippocampal, and spinal neurons, NMDA (1-300 µM) and glycine (10 nM-10 µM) induced currents in a concentration-dependent manner only in the presence of both agonists. Currents were characterized by the presence of two phases: a transient and a sustained phase (Fig. 3A-C). NMDA- and glycine-activated currents were measured at steady state and data were transformed into CRC. The agonist pEC₅₀ and slope values are reported in Table 2.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Traces recording NMDA- (100 µM), glycine (3 µM)-induced currents from rat cortical (A), spinal cord (B), and hippocampal (C) neurons from embryonic rat. The concentrations of GV196771A are described in the figure.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   A, inhibitory effect of GV196771A in rat cortical (), spinal cord (), and hippocampal () neurons from embryonic rat. B, CRC to a maximal concentration of NMDA (100 µM) in the presence of increasing concentrations of glycine. C, CRC to a maximal concentration of glycine (10 µM) in the presence of increasing concentration of NMDA. GV196771A at 1 µM () antagonized in a competitive manner the glycine CRC (; 4B) and in a noncompetitive way the NMDA CRC (; 4C).

Suppression of Spinal Cord Wind-Up by GV196771A. A significant reduction (p < .01) of the AUC "wind-up" response was observed after 60 min of superfusion of the isolated spinal cords with GV196771A at 10 µM (Fig. 5A; n = 6); the "wind-up" was depressed by 25.5 ± 2% with respect to controls. In parallel experiments, we confirmed that in the same preparation morphine (n = 2) or D-AP5 (n = 3) suppressed the "wind-up" (Fig. 5, B and C).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Suppressions of spinal cord "wind-up" by GV196771A, D-AP5, and morphine. A, effect of 1-h superfusion with GV196771A (10 µM) dissolved in DMSO containing (0.1%) Krebs' solution. B, effect of a 20-min superfusion with D-AP5 (50 µM). C, effect of a 30-min superfusion with morphine (3 µM). Each panel shows two superimposed voltage records obtained from the same preparation before (CTRL) and during superfusion of the compounds indicated.

Antihyperalgesic Activity of GV196771A

GV196771A Reduces Thermal Hyperalgesia in CCI Model. Prophylactic treatment. Before surgery, no significant difference between left and right thermally induced paw withdrawal latencies was detected in both vehicle- (mean ± S.E.M.: 10.56 ± 0.51 s versus 10.47 ± 0.52 s) and GV196771A- (mean ± S.E.M.: 10.60 ± 0.36 s versus 10.48 ± 0.32 s) treated groups.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   A, graphs illustrate the time course of thermal hyperalgesia after sciatic nerve ligation. In the vehicle-treated group (; n = 8), DSs from days 14 to 30 after surgery were negative and significantly different with respect to the preoperative value (day 0; *p < .05 versus basal value). In GV196771A-treated group (3 mg/kg p.o.; ; n = 9) no significant variation in DSs was observed during the whole experiment. Moreover, in GV196771A-treated animals, DSs were significantly lower on days 14 to 21 compared with vehicle-treated animals (p < .05 versus vehicle-treated group). B, time course of the thermal hyperalgesia in the nonligated right paws. The graphs illustrate withdrawal latencies of the nonligated paws in prophylactically treated animals; no significant variations in both vehicle (; n = 8) and GV196771A groups (3 mg/kg p.o.; ; n = 9) with respect to the basal values (day 0) were observed during the whole experiment. C, GV196771A produces a decrease of thermal paw withdrawal latency in the therapeutic treatment. Control (), GV196771A 1 (), 3 (), and 10 mg/kg, p.o. (). GV196771A at 3 and 10 mg/kg reverses the thermal hyperalgesia at 1, 4, and 8 h after drug treatment (*p < .05; n = 6-16 for each group).

Therapeutic treatment. GV196771A (1-10 mg/kg p.o.) produced a dose-related reversal of thermal hyperalgesia increasing the latency of the ligated paw. At 3 and 10 mg/kg the reduction of thermal hyperalgesia was significant (p < .05), whereas it was not at 1 mg/kg p.o. (Fig. 6C and Table 3). By measuring the analgesic effect at 1 h after treatment, an ED₅₀ of 2.95 (1.50-8.44) mg/kg was calculated.

GV196771A Reduces Pain Behavior in Mice Paw Formalin Test: Acute Administration. In the vehicle-treated groups, s.c. injection of formalin induced marked spontaneous nociceptive behaviors. The values of total licking time measured during the EP and the LP were 125.5 ± 7.2 and 317.3 ± 42.8 s, respectively, in the methocel-treated group (Fig. 7A) and 148.8 ± 12.1 and 529.9 ± 46.0 s, respectively, in the saline-treated group (Fig. 7B).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   A GV196771A (0.1-10 mg/kg p.o.) produces inhibition of the LP of the formalin test in mice. n = 7-19 for each group. *p < .05 versus vehicle. B, morphine (0.3-10 mg/kg p.o.) produces inhibition of both EP and LP of the formalin test in mice. n = 8-10 for each group. *p < .05 versus vehicle.

Chronic Treatment with GV196771A Did Not Induce Tolerance in Mice Paw Formalin Test. In g2, s.c. injection of formalin at day 9 induced marked spontaneous nociceptive behaviors. The licking time values measured during the EP and the LP were 125.1 ± 11.9 and 555.4 ± 147.1 s, respectively (Fig. 8A).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Morphine, but not GV196771A, induces tolerance. A, in control group (g2) s.c. injection of formalin induced marked nociceptive behaviors. GV196771A 3 mg/kg p.o. administered at day 9, after chronic treatment with methocel (g5), had no effect on the EP. On the contrary, a significant reduction of the hyperalgesic responses in LP was observed. When GV196771A 3 mg/kg p.o. was administered at day 9, after chronic treatment with GV196771A 3 and 10 mg/kg p.o. for 8 days the antihyperalgesic activity of the compound was maintained in the LP (g6 and g7, respectively). n = 10-21 for each group. *p < .05 versus g2. B, in the vehicle-treated group (g1), s.c. injection of formalin induced marked nociceptive behaviors. Animals receiving morphine 3 mg/kg i.p. at day 9, after chronic treatment with saline (g3), showed significant attenuation on basal nociceptive responses in both phases. Administration of morphine 3 mg/kg i.p. at day 9, after chronic morphine treatment (10 mg/kg i.p.; g4), was ineffective in both phases. n = 10-21 for each group. *p < .05 versus g1.

Chronic Treatment with Morphine-Induced Tolerance in Mice Paw Formalin Test. In g1 s.c. injection of formalin at day 9 induced marked spontaneous nociceptive behaviors. The licking time values measured during the EP and the LP were 119.4 ± 20.5 and 494.6 ± 92.1 s, respectively (Fig. 8B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Among the different classes of NMDA receptor antagonists, ligands at the glycine site have recently been reported to elicit antinociception against prolonged noxious stimulation in the absence of a marked influence upon motor coordination (Millian and Seguin, 1994). The results obtained with GV196771A further support the role of NMDA receptor in the development and maintenance of chronic pain and the analgesic effect of the NMDA glycine site antagonists.

Inhibition of NMDA Receptors. GV196771A was found to possess a high affinity (pK_i value of 7.56) for the glycine site of the NMDA receptors in the rat cerebral cortex. This value was in agreement with that found in functional experiments carried out with [³H]TCP. As expected, [³H]TCP could bind to the NMDA channel only when the receptor is activated by the simultaneous presence of the two agonists, NMDA and glycine. In these experiments GV196771A showed a competitive behavior displacing in a parallel manner the [³H]TCP binding induced by glycine CRC with an apparent pK_B of 7.13. On the other hand GV196771A was a very week ligand for the AMPA and kainate receptors and for the glutamate binding site of the NMDA receptor.

Electrophysiological Recordings: Patch-Clamp in Primary Cultures and Wind-Up in Baby Rat Spinal Cord. The binding results were confirmed and extended by further functional studies using the patch-clamp technique in embryonic rat neurons taking from cortex, hippocampal, and spinal cord. In these functional preparations, the simultaneous presence of NMDA and glycine induced currents flowing through the NMDA receptor channel. GV196771A antagonized the NMDA glycine-induced currents in all three preparations, although in the spinal cord neurons the pK_B value was higher (8.05) in respect to those found in cortical (7.47) and hippocampal neurons (7.86). Also in this study, GV196771A was found to be a competitive antagonist of the glycine-induced currents whereas the antagonism of GV196771A on NMDA-induced currents was not reversed by increasing the agonist concentration.

Antihyperalgesic Activity. We investigated the effects of GV196771A in a rat model of painful mononeuropathy (CCI) and in the formalin test in mice.

Tolerance Study. The results reported here indicate that GV196771A, different from opioids, does not induce tolerance of its antihyperalgesic activity after 8 days of chronic treatment.