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BEHAVIORAL PHARMACOLOGY
-Hydroxybutyrate: Role of Training Dose
Departments of Psychiatry and Pharmacology (W.K., C.P.F.), University of Texas Health Science Center at San Antonio, San Antonio, Texas; and Department of Pharmaceutical Sciences (W.C., S.L.M., A.C.), University of Maryland, Baltimore, Maryland
Received October 10, 2005; accepted December 2, 2005.
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Abstract |
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-Hydroxybutyrate (GHB) is a drug of abuse with actions at GHB and GABA receptors. This study examined whether the relative importance of GABAA, GABAB, and GHB receptors in the discriminative stimulus effects of GHB depends on the training dose. In comparison with a previous 100 mg/kg GHB-saline discrimination, pigeons were trained to discriminate either 178 or 56 mg/kg GHB from saline. Increasing the training dose shifted the GHB gradient to the right, and decreasing it shifted the gradient to the left. Similar shifts occurred with the GHB precursor -butyrolactone, which substituted for GHB, and with the GABAB agonists baclofen and 3-aminopropyl(methyl)phosphinic acid hydrochloride (SKF97541) and the benzodiazepine diazepam, each of which produced at most 54 to 68% GHB-appropriate responding. The benzodiazepine antagonist flumazenil, the benzodiazepine inverse agonist ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-
]-[1,4]-benzodiazepine-3-carboxylate (Ro 15-4513), and the GHB receptor antagonist (2E)-5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene ethanoic acid (NCS-382) produced a maximum of 66 to 97% GHB-appropriate responding in animals discriminating 56 or 100 mg/kg GHB and a maximum of 1 to 49% in animals discriminating 178 mg/kg. NCS-382 did not attenuate the effects of GHB. The GABAB antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348) blocked GHB at all training doses. The results suggest that increasing the training dose of GHB increases the pharmacological selectivity of its discriminative stimulus effects. At a high training dose, diazepam-insensitive GABAA receptors, for which flumazenil and Ro 15-4513 have affinity, may no longer be involved. Diazepam-sensitive GABAA receptors and GABAB receptors appear to play a similar role at all training doses. There was no evidence for GHB receptor involvement.
-Hydroxybutyrate (GHB) is used to treat narcolepsy but is also a drug of abuse, as are some of its precursors (for review, see Nicholson and Balster, 2001
). GHB occurs naturally in the brain, where it may exert its effects through specific GHB receptors, through GABAB receptors (Lingenhoehl et al., 1999), and, because it is metabolically converted to GABA (Vayer et al., 1985), indirectly through all GABA receptor subtypes. The precise roles of GHB in central nervous system function, however, are not well understood.
Drug discrimination has proved useful to study mechanisms of drug action because it can provide sensitive and pharmacologically selective assays of in vivo effects (e.g., Colpaert and Balster, 1988). Rats can discriminate GHB from saline (Winter, 1981; Colombo et al., 1995a,b, 1998; Metcalf et al., 2001; Carter et al., 2003; Baker et al., 2004), and the discriminative stimulus effects are selective because pharmacologically unrelated drugs (e.g., phencyclidine, ketamine) do not substitute for GHB (Winter, 1981; Carter et al., 2003; Baker et al., 2004). In addition, GHB does not substitute for training drugs that are pharmacologically unrelated to GHB (Beardsley et al., 1996; Woolverton et al., 1999). Together, these studies suggest that the discriminative stimulus effects of GHB involve multiple mechanisms, with a prominent role for GABAB receptors, a less important role for GABAA receptors, and a role for GHB receptors that has not been clearly delineated.
The discriminative stimulus effects of ethanol, like those of GHB, likely involve multiple mechanisms (e.g., Grant, 1994; Kostowski and Bienkowski, 1999), with the GABAA and N-methyl-D-aspartate (NMDA) receptor complexes being particularly important. The relative prominence of the GABAA and the NMDA components appears to depend on the training dose, with a greater role for GABAA at lower ethanol training doses and a greater role of NMDA at higher ethanol training doses (for review, see Grant, 1999). The prominence of the different receptor systems involved in the discriminative stimulus effects of GHB may also depend on the training dose. In rats trained to discriminate either 300 or 700 mg/kg GHB (i.g.) from water, diazepam reportedly produced more GHB-appropriate responding in the low than in the high training dose group, the GABAB agonist baclofen was less potent to produce GHB-appropriate responding in the low than in the high training dose group, and the GABAB antagonist CGP35348 attenuated the discriminative stimulus effects of the low training dose less than those of the high training dose (Colombo et al., 1998). These findings have been taken to suggest a more important role of GABAA receptors at low doses and of GABAB receptors at higher GHB training doses (Colombo et al., 1998). The present study is part of an effort to further examine this hypothesis.
Recently, we reported that pigeons can reliably discriminate GHB from vehicle and that their sensitivity to the discriminative stimulus and response rate-decreasing effects of GHB does not seem to differ markedly from that of rats (Koek et al., 2004). There were, however, some apparent differences between the results obtained in pigeons and in rats. For example, the GABAB receptor agonist baclofen was less potent and effective to produce GHB-like discriminative stimulus effects in pigeons than in rats, and the GABAB receptor antagonist CGP35348 was less potent and effective to antagonize the discriminative stimulus effects of GHB (Carter et al., 2003; Koek et al., 2004). These findings suggest a possible species difference in the extent to which GABAB receptors are involved in the discriminative stimulus effects of GHB. The relative importance of GABAB mechanisms, however, may also be influenced by the training dose of GHB (Colombo et al., 1998). Varying the training dose in both species will help to identify the extent to which the discriminative stimulus effects are species-dependent.
In comparison with the previous 100 mg/kg GHB (i.m.)-saline discrimination (Koek et al., 2004), different groups of pigeons were trained to discriminate either a lower or a higher dose of GHB from saline. The following compounds were examined in all three groups: GHB and its metabolic precursor, -butyrolactone (GBL); putative GHB receptor antagonist NCS-382; GABAB receptor agonists baclofen and SKF97541; GABAB receptor antagonist CGP35348; GABAA receptor agonist THIP; positive GABAA modulators diazepam and pentobarbital; benzodiazepine antagonist flumazenil and inverse agonist Ro 15-4513, which were found previously to substitute for 100 mg/kg GHB in pigeons (Koek et al., 2004); dopamine D2/D3 receptor antagonist haloperidol; and compounds pharmacologically unrelated to GHB, i.e., cocaine, ketamine, and morphine. The disulfide-reducing agent dithiothreitol (DTT), which reportedly blocks the discriminative stimulus effects of GHB by suppressing GABAB receptor function (Carai et al., 2004), was examined only in the 100 and 178 mg/kg GHB training dose groups.
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Materials and Methods |
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Apparatus. Experiments were conducted in sound-attenuating, ventilated chambers (BRS/LVE, Laurel, MD) equipped with two response keys that could be illuminated by red lights. After completion of each fixed ratio, the key lights were extinguished for 4 s, during which time a white light illuminated the hopper where food (Purina Pigeon Checkers, St. Louis, MO) was available. Chambers were connected by an interface (MED Associates Inc., St. Albans, VT) to a computer that used MED-PC IV software (MED Associates Inc.) to monitor and control inputs and outputs and to record the data.
Procedure. Three different groups of pigeons were trained to discriminate between saline and a different training dose of GHB. Previously, six pigeons had been trained to discriminate 100 mg/kg GHB from saline (Koek et al., 2004); here, in comparison, lower (i.e., 32 mg/kg) and higher (i.e., 320 mg/kg) training doses were used in two different groups of nine pigeons. After 50 training sessions, fewer than one-half of the animals that received 32 mg/kg and fewer than one-half of the animals that received 320 mg/kg met criterion (see Results). Therefore, the lower training dose was increased to 56 mg/kg, and the higher training dose was decreased to 178 mg/kg. The discrimination training and testing procedure was similar to that described in detail elsewhere (Koek et al., 2004). Briefly, before each daily session, subjects received either GHB or saline (i.m.) and were immediately placed into the chamber; drug and vehicle training sessions occurred with equal frequency. Sessions started with a period of 15 min, during which the lights were off, and key pecks had no programmed consequence. Subsequently, the left and the right keys were illuminated red, and 20 consecutive responses on the injection appropriate key resulted in 4-s access to food (for half of the pigeons, the left key was active after GHB, and the right key was active after saline, and for the other half, the left key was active after saline and the right key after GHB). Responses on the incorrect key reset the fixed ratio requirement on the correct key. The response period ended after 30 food presentations or 15 min, whichever occurred first. Initially, pigeons had to satisfy the following criteria for at least seven of nine consecutive sessions: 
90% of the total responses on the correct key and fewer than 20 responses on the incorrect key before the first food presentation. Thereafter, tests were conducted when these criteria were satisfied during two consecutive (drug and saline) training sessions. Test sessions were the same as training sessions (i.e., a 15-min period, followed by a response period that ended after 30 food presentations or 15 min, whichever occurred first), except that food was available after completion of 20 consecutive responses on either key. Drug (or vehicle) treatments were given immediately before the session, and pretreatments were given 10 min before treatments.
Data Analysis. The mean percentage of responses on the GHB key ± 1 S.E.M. and the mean rate of responding (expressed as a percentage of the saline control rate) ± 1 S.E.M. during test sessions were plotted as a function of dose. When an animal responded at a rate less than 20% of the saline control rate, discrimination data from that test were not included in the average. Mean percentages of responses on the GHB key values were calculated only when they were based on at least one-half of the animals tested.
Differences among the dose-response curves in the different training dose groups to produce GHB-appropriate responding and to decrease response rate were analyzed by simultaneously fitting straight lines to the individual dose-response data by means of GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego CA), using the following equation: effect = slope x log(dose) + intercept. Straight lines were fitted to the linear portion of dose-response curves, which comprised data points at doses with effects immediately below and above 50% and included not more than one dose with >75% effect and not more than one dose with < 25% effect. Dose-response curves that failed to cross the 50% level were not analyzed by linear curve fitting. Differences between individual means were analyzed by a randomization test for dependent samples (Siegel and Castellan, 1988; Edgington, 1995).
An important feature of the GraphPad program is the possibility to fit models of varying complexity to the data. With GraphPad, a model can be simplified by selecting common parameters (e.g., common slope, common intercept) and to compare simpler models with more complex models by means of an F ratio test. If the calculated F for two models is statistically significant, this indicates that the more complex model is required to fit the data. However, if the value of F is not significant, the simple model should be used (for detailed examples of this approach; see Kenakin, 1997). For best-fitting models of dose-response curves that crossed the 75% level of effect, doses corresponding to the 50% level of effect (D50), potency ratios, and their 95% confidence limits were calculated by parallel line analysis (Tallarida, 2000) of data from individual subjects. The slope values calculated by GraphPad Prism were used to constrain the fit of the parallel line assay.
Drugs. Drugs were dissolved in sterile water or saline, unless otherwise noted, and included flumazenil and Ro 15-4513 (gifts from F. Hoffmann LaRoche Ltd., Basel, Switzerland), GHB, GBL, (±)-baclofen, diazepam, haloperidol, DTT, and pentobarbital sodium (Sigma-Aldrich Corp., St. Louis, MO); NCS-382 (sodium salt), cocaine hydrochloride, and morphine sulfate (National Institute on Drug Abuse, Research Technology Branch, Rockville, MD); THIP hydrochloride (gaboxadol) (Tocris Cookson Inc., Ellisville, MO); ketamine hydrochloride (Ketaset; Fort Dodge Laboratories Inc., Fort Dodge, IA); and CGP35348 (sodium salt) and SKF97541 hydrochloride (synthesized at the University of Maryland, Baltimore, MD). Diazepam was dissolved at different concentrations in the same vehicle consisting of 70% Emulphor, 20% sterile water, and 10% ethanol (by volume). Flumazenil was dissolved at different concentrations in the same vehicle consisting of 50% saline, 40% propylene glycol, and 10% ethanol (by volume). All compounds were injected i.m. in a volume of 0.1 to 1.0 ml. Doses are expressed as the form of the drug listed above.
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Results |
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Under test conditions, GHB dose-dependently increased responding on the GHB-appropriate key (Fig. 1, top left panel) and decreased response rate (bottom left panel) in all three training dose groups. Results obtained in tests of saline did not differ significantly among the groups (randomization test) [mean response rate (±S.E.M.), 1.66 (0.09) responses(s)]. Linear curves fitted to the dose-response data obtained with GHB in each of the groups had slope values significantly different from zero, indicating that GHB significantly increased GHB-appropriate responding and decreased response rate in each group. The simplest model that could be fitted to the GHB discrimination data was one with a common slope value for the 56 and 100 mg/kg groups, an individual slope value for the 178 mg/kg group, and individual intercept values for all three groups, indicating that as the training dose increased the dose-response curve significantly shifted to the right and became steeper. Increasing the training dose increased GHB's D50 to produce GHB-appropriate responding (Table 1). The effects of GHB on response rate, which could be fitted with a common slope value, appeared to differ among the groups: GHB was significantly less potent to decrease response rate in the 178 mg/kg group than in the other groups (Table 1).
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GBL increased responding on the GHB-appropriate key in all three groups (Fig. 1, top middle panel) to a maximum similar to that obtained with GHB. As with GHB, the dose-response curves obtained with the GHB precursor GBL were shifted to the right at higher training doses (Fig. 1, middle panels). The discrimination data obtained with GBL could be fitted with a common slope but required three different intercept values, indicating the dose-response curves to be significantly shifted to the right in a parallel manner (Table 1). The response rate data obtained with GBL could be fitted with a common slope only for the 56 and 178 mg/kg groups; GBL was significantly less potent to decrease response rate in the 178 mg/kg group than in the 56 mg/kg group. GBL decreased response rate in the 100 mg/kg group along a dose-response curve shallower than and in between those obtained in the other two groups.
NCS-382 increased responding on the GHB-appropriate key in the 56 and 100 mg/kg groups (to a maximum of 67 and 70%, respectively), but not in the 178 mg/kg group (maximum of 1%) (Fig. 1, top right panel). The discrimination data obtained in the 56 and 100 mg/kg groups could be fitted with a common slope and intercept, indicating that NCS-382 was equipotent to produce GHB-appropriate responding in these two groups. NCS-382 significantly decreased response rate only at a dose of 560 mg/kg (Fig. 1, bottom right panel). When given before the training dose of GHB in the 178 mg/kg group, NCS-382 did not decrease the percentage of responses on the GHB-appropriate key (Fig. 1, top right panel), but its potency to decrease response rate (Fig. 1, bottom right panel) appeared to be enhanced. This enhancement was statistically significant because the simplest model that fitted the response rate data obtained with NCS-382 in the 178 mg/kg group had a common slope but significantly different intercepts. In the presence of GHB, the D50 of NCS-382 to decrease response rate was 290 (180–480) mg/kg.
Baclofen dose-dependently increased the percentage responses on the GHB-appropriate key in each of the three groups to a maximum ranging from 60 to 66% (Fig. 2, top left panel). The discrimination data could be fitted with a common slope but with three significantly different intercepts, indicating that baclofen was significantly less potent to produce GHB-appropriate responding at higher training doses. Baclofen decreased response rate similarly in all three groups (Fig. 2, bottom left panel; Table 1). Like baclofen, SKF97541 increased the percentage responses on the GHB-appropriate key in each of the three groups, to maxima ranging from 38 to 68% (Fig. 2, top middle panel). The discrimination data obtained with SKF97541 appeared similar to those of baclofen, with increasing training doses associated with rightward shifts of the dose-response curves. SKF97541 was significantly less potent to decrease response rate in the 178 mg/kg group than in the other groups (Fig. 2, bottom middle panel; Table 1). GP35348 dose-dependently decreased the percentage responses on the GHB-appropriate key in all three groups (to values ranging from 23 to 9%; Fig. 2, top right panel). CGP35348 did so significantly more potently in the 56 mg/kg group than in the other two groups (Table 1). The response rate was not significantly affected by CGP35348 combined with the training dose of GHB (Fig. 2, bottom right panel) (randomization test).
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The barbiturate pentobarbital and the GABAA receptor agonist THIP increased responding on the GHB key similarly in all three groups (Fig. 4, top left and middle panels) to maxima between 28 and 49% and decreased response rate similarly in each group (Fig. 4, bottom left and middle panels; Table 1). The dopamine D2/D3 receptor antagonist haloperidol increased the percentage of responses on the GHB-appropriate key in the 56 and 100 mg/kg groups (to a maximum of 52 and 34%, respectively) but not in the 178 mg/kg group (Fig. 4, top right panel). Haloperidol appeared to produce more GHB-appropriate responding in the 56 mg/kg group than in the other groups. The results in the 56 mg/kg group differed significantly (P < 0.05, randomization test) from those in the 100 mg/kg group at 0.1 mg/kg haloperidol and differed significantly (P < 0.05) from those in the 178 mg/kg group at 1 mg/kg haloperidol. Haloperidol decreased response rate similarly in all three groups (i.e., common slope and intercept) (Fig. 4, bottom right panel). When haloperidol was given before the training dose of GHB in the 178 mg/kg group, the minimum percentage responses on the GHB-appropriate key was 90 (Fig. 4, top right panel), and the potency of haloperidol to decrease response rate (Fig. 4, bottom right panel) did not significantly change in the presence of GHB (i.e., the dose-response curves of haloperidol to decrease response rate with or without GHB present could be fitted with a common slope and intercept).
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When tested up to and including response rate-decreasing doses in the 100 and 178 mg/kg groups, the maximum percentage of GHB-appropriate responding observed with DTT was 5.9% (Table 2). DTT did not significantly decrease the percentage of GHB-appropriate responding observed with the training dose of GHB and did not significantly attenuate the response rate-decreasing effects of baclofen and SKF97541 in the 100 mg/kg group.
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Discussion |
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; Koek and Slangen, 1982). Similar shifts were observed with the GHB precursor GBL. Increasing the training dose shifted the dose-response curves of their rate-decreasing effects to the right, consistent with findings that the effects of GHB on response rate show tolerance (Eckermann et al., 2004).
NCS-382 produced GHB-appropriate responding when the training dose was low or medium but not when it was high. Under these latter conditions, NCS-382 failed to attenuate the discriminative stimulus effects of GHB. NCS-382 antagonized the discriminative stimulus effects of GHB in some studies (Colombo et al., 1995a; Baker et al., 2005) and not in others (Carter et al., 2003; Koek et al., 2005); it has been suggested that procedural factors, such as the pretreatment time, may be responsible (Baker et al., 2005). The observation that NCS-382 shares effects with GHB is consistent with reports of partial GHB-like effects of NCS-382 in GHB-discriminating rats (Carter et al., 2003) and of GHB-induced loss of righting enhanced by NCS-382 (Carai et al., 2001). Agonist actions at GABAB receptors may be involved in the GHB-like effects of NCS-382 (Carai et al., 2002; Koek et al., 2004). Clearly, selective GHB receptor antagonists that lack GHB-like effects would greatly facilitate the study of drug actions at specific GHB receptors.
The dose-response curves for the discriminative stimulus effects of the GABAB receptor agonists baclofen and SKF975412, like those of GHB and GBL, were shifted to the right at higher training doses. In the high training dose group, the response rate decreasing effects of SKF97541, but not those of baclofen, showed tolerance. The maximal level of GHB-appropriate responding observed with baclofen and SKF97541 was partial and did not vary markedly with the training dose. Thus, the finding that baclofen substitutes partially in 100 mg/kg GHB-trained pigeons (Koek et al., 2004) holds also for another GABAB agonist and across a range of training doses. Conversely, in rats, baclofen substituted completely for GHB (Colombo et al., 1998). Although baclofen appears to produce more GHB-appropriate responding in rats (Colombo et al., 1998; Carter et al., 2003) than in pigeons, the discriminative stimulus effects of baclofen and GHB are not identical in rats because rats can discriminate GHB from baclofen (Koek et al., 2005). The finding that baclofen and SKF97541 substitute only partially for GHB in pigeons suggests that agonist actions at GABAB receptors alone are not sufficient to account for the discriminative stimulus effects of GHB in this species. That GABAB receptors are nevertheless involved is suggested by the attenuation of the discriminate stimulus effects of GHB by the GABAB receptor antagonist CGP35348 in all three training dose groups. The antagonism appeared to be less complete at the low than at the high training dose, consistent with previous findings in rats (Colombo et al., 1998). Irrespective of the training dose, CGP35348 maximally antagonized the discriminative stimulus effects of GHB only at doses (i.e., 320–560 mg/kg) 6- to 10-fold higher than the dose needed to completely block GHB-appropriate responding produced by baclofen in 100 mg/kg GHB-discriminating pigeons (i.e., 56 mg/kg; Koek et al., 2004). There is increasing evidence for the existence of functional GABAB receptor subtypes (e.g., Seabrook et al., 1990; Bonanno and Raiteri, 1993; Yamada et al., 1999). Thus, this differential antagonism of GHB and baclofen by CGP35348, which recently has been observed also in rats (L. P. Carter, W. Chen, A. Koop, W. Koek, and C. P. France, unpublished observations), is consistent with the possible involvement of different GABAB receptor subtypes in the discriminative stimulus effects of GHB and baclofen. Whichever GABAB receptor subtypes will be found to be involved, the present results suggest that the extent to which they are involved in pigeons is not markedly affected by the training dose of GHB.
The dose-response curve for the discriminative stimulus effects of diazepam shifted to the right with increasing training dose, whereas its maximum effect did not vary markedly (i.e., with not more than 13%) and remained partial (i.e., between 41 and 54%). The latter observation differs from the finding in GHB-trained rats that diazepam produced almost 70% GHB-appropriate responding in the low training dose group but less than 20% in the high training dose group (Colombo et al., 1998). Possible species differences and procedural factors (e.g., route of administration) may underlie this discrepancy. The results obtained here with the GABAA receptor-positive modulators diazepam and pentobarbital and with the GABAA receptor agonist THIP do not support the hypothesis that the role of GABAA receptors is more important in the discriminative stimulus effects of GHB at lower training doses.
Training dose, however, may affect the role of a particular subtype of GABAA receptor in the discriminative stimulus effects of GHB. Flumazenil produced more than 50% GHB-appropriate responding in the low and intermediate training dose groups. The discriminative stimulus effects of low doses of flumazenil in pigeons are likely mediated by diazepam-insensitive GABAA receptors (Wong et al., 1993; Acri et al., 1995, 1997). A role for these receptors in the effects examined here is supported by the observation that Ro 15-4513, which like flumazenil has high affinity for diazepam-insensitive GABAA receptors, had effects similar to those of flumazenil, i.e., more than 50% GHB-appropriate responding in the low and intermediate training dose group. It is unclear why the maximal effects of flumazenil and Ro 15-4513 appeared to be smaller in the low than in the intermediate training dose group. For flumazenil, this may be related to more variation between animals of the dose producing maximal effects and for Ro 15-4513 to larger rate-decreasing effects in the low training dose group. Nevertheless, both compounds produced marked GHB-appropriate responding at the low and the intermediate training dose, suggesting a role for diazepam-insensitive GABAA receptors in the discriminative stimulus effects of these doses of GHB. At a high training dose, however, the maximal effects of flumazenil and Ro 15-4513 were markedly lower, and flumazenil had neither agonist nor antagonist activity, suggesting a diminished role for diazepam-insensitive GABAA receptors in the discriminative stimulus effects of a high training dose of GHB.
Increasing the training dose generally produced a left and downward shift of the dose-response curves of haloperidol, cocaine, and morphine to produce GHB-appropriate responding. Increased pharmacological selectivity at higher training doses is consistent with results of other studies in which the training dose was varied (e.g., White and Appel, 1982; Picker et al., 1990; Mansbach and Balster, 1991). The lack of substantial GHB-appropriate responding produced by haloperidol, cocaine, and morphine at intermediate and high training doses confirms and extends results obtained in GHB-discriminating rats (Winter, 1981; Carter et al., 2003; Koek et al., 2005) and further suggests that the discriminative stimulus effects of GHB do not primarily involve modulation of dopamine or opioid systems.
The uncompetitive, ion channel-blocking NMDA antagonist dizocilpine produces intermediate levels of drug-appropriate responding in various discriminations with training drugs pharmacologically unrelated to dizocilpine (Koek et al., 1995). This intermediate responding may not reflect shared discriminative stimulus effects but a disruption of discriminative responding, conceivably related to the ability of NMDA antagonists to produce state dependence (Jackson et al., 1992). Such disruptions occur also with other uncompetitive, ion channel-blocking NMDA antagonists, such as phencyclidine and ketamine (Koek et al., 1993). In morphine-discriminating pigeons, ketamine produced intermediate responding that did not markedly change when the training dose was varied, a finding that is consistent with a general disruption of discriminative responding (Koek and Woods, 1989). Here, ketamine also produced intermediate responding, but its effects changed as a function of training dose. At lower training doses, ketamine's dose-response curve for GHB-appropriate responding shifted to the left, but its maximum did not appear to change. Although this may indicate that ketamine produced these effects because it shared discriminative stimulus effects with GHB, it is also possible that ketamine was more potent to disrupt discriminative responding at lower training doses. If the effects of ketamine observed here are related to its disruption of discriminative responding, one would expect ketamine to produce intermediate responding not only when given alone but also when given before the training dose. When given before the training dose, however, ketamine did not markedly decrease GHB-appropriate responding, consistent with the possibility that ketamine shares discriminative stimulus effects with GHB. Examining ketamine-GHB interactions in more detail may help to further test this possibility.
DTT is thought to alter the structural stability of GABAB receptors and reportedly antagonized GABAB agonist- and GHB-induced sedation/hypnosis in mice and completely blocked the discriminative stimulus effects of GHB in rats (Carai et al., 2004). DTT only partially attenuated the discriminative stimulus effects of GHB in rats (Koek et al., 2005), and the present results show that DTT also failed to block the discriminative stimulus effects of GHB in pigeons. Thus, the generality of the GHB-blocking effects of DTT appears to be limited.
In summary, the results suggest that increasing the training dose of GHB increases the pharmacological selectivity of its discriminative stimulus effects. At a high training dose, diazepam-insensitive GABAA receptors are no longer involved in the discriminative stimulus effects of GHB in pigeons. Whether these receptors are involved in the discriminative stimulus effects of GHB in rats awaits further studies with different training doses. Varying the training dose did not appear to change the extent of GABAB and diazepam-sensitive GABAA receptor involvement, which may be more limited in the discriminative stimulus effects of GHB in pigeons than in rats. Different GABAB receptor subtypes may account for some of the differences between GHB and GABAB receptor agonists.
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ABBREVIATIONS: GHB, -hydroxybutyrate; N-methyl-D-aspartate; CGP35348, 3-aminopropyl(diethoxymethyl)phosphinic acid; GBL, -butyrolactone; NCS-382, (2E)-5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene ethanoic acid; SKF97541, 3-aminopropyl(methyl)phosphinic acid hydrochloride; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]epyridine-3-ol; Ro 15-4513, ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-]-[1,4]-benzodiazepine-3-carboxylate; DTT, dithiothreitol.
Address correspondence to: Dr. Wouter Koek, Departments of Psychiatry and Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code 7792, San Antonio, TX 78229-3900. E-mail: koek{at}uthscsa.edu
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