Behavioral effects of benzodiazepine ligands in non-dependent, diazepam-dependent and diazepam-withdrawn baboons

European Journal ofl’hartnacology.

202 (1991)

159-169

159

1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ADONIS 001429999100582W 8

EJP 52023

Christine A. Sannerud ’

Deparmtent

of Psychiatry and Behavioral

Ac~enrte, Traylor 624. Baltimore,

I,*, Michael Allen 3, James M. Cook 3 and Roland R. Griffiths I32 Sciences, ’ Department

of Neuroscience.

MD 21205, U.S.A. and J Department

Received 4

The Johns Hopkins Uttirersity School of Medicine. 720 Rut/and

of Chmistry,

Vnit’ersil)’ of Wisconsin, Mihvattkee,

WI 53201, V.S.A.

June 1991,accepted 25 June 1991

Acute i.m. injections of benzodiazepine receptor liyands were administered to baboons before l-h observational sessions. The agonist midazolam produced sedative effects, the antagonist flumazenil produced no behavioral effects, the inverse agonist FG’142 produced tremor and the inverse agonist 3-carboethoxy-p-carboline hydrochloride (PCCE) produced tremor. vomiting. jerks and seizures. Co-administration of these drugs (midazolam + PCCE. midazolam + flumazcnil or flumazcnil + PCCE) produced a mutual antagonism of these effects. Compared to the non-dependent condition. in the diazepam-dependent condition (baboons maintained on 20 mg/kg per day diazepam) and withdrawn condition (dependent baboons tested during withdrawal), midazolam produced decreased sedative effects, flumazenil produced increased effects (i.e., tremor, vomiting and jerks), and pCCE produced increased frequency of seizures. Taken together, these data suggest that (1) benzodiazcpine rcccptor ligands lie on a continuum of behavioral activity, and (2) chronic diazepam administration alters the behavioral effects of these benzodiazepine ligands, producing a shift in the direction of the inverse agonist. Diazepam; P-CCE (3-carboethoxy-p-carboline hydrochloride); FG7142; Flumazcnil; Midazolam; Dependence; GABA (y-aminobutyric acid); (Baboon)

1. Introduction

The discovery of the pharmacologically unique GABA/benzodiazepine receptor complex that mediates bidirectional effects of different drugs has led to new concepts of receptor functioning and drug action. The benzodiazepine receptor is thought to be a modulatory site coupled to the GABA, receptor-chloride channel that functions as a secondary receptor affecting ligand binding and/or channel gating of the primary GABA receptor (Haefely, 1990). Three classes of receptor ligands (agonists, antagonists and inverse agonists) that bind with qigh affinity to the GABA/benzodiazepine receptor system have been shown to differentially modulate the effects mediated by GABA, producing a spectrum of biochemical and behavioral effects (cf. Richards et al., 1986; Bracstrup et al , 1983; Pole et al., 1982) Within this theoretical framework, benzodiazepine receptor agonists can be considered to Correspondence to: CA. Sannerud. Department of PsychiW, 623 Traylor Building, 720 Rutland Avenue, The Johns Hopkins University Medical School, Baltimore. MD 21205. U.S.A. d: Currcnl affiliation: NIDA Addiction Rcscarch Center. Baltimore. MD. U.S.A.

have positive intrinsic activity: they have anticonvulsant, anxiolytic and sedative properties, and enhance the effects of GABA (y-aminobutyric acid). Inverse agonists have negative intrinsic activity: they produce effects that are mirror images of those produced by the benzodiazepine agonists (e.g. anxiogenic and convulsant and reduce the effects of GABA in viva). Antagonists have little or no intrinsic activity: they bind to the receptor and block the receptor-mediated behavioral and pharmacological effects of both agonists and inverse agonists. Chronic administration of bcnzodiazepine agonists may produce functional changes at the benzodiazepine receptor that result in alterations in the behavioral effects of the benzodiazepine receptor ligands. It is well established that chronic treatment with benzodiazepine agonists results in tolerance to the sedative or anticonvulsant effects of the drug (File, 1985; Gonsalves and Gallager, 1985, 1987; Petersen and Jensen, 1987a, b; Tietz and Rosenberg, 1988; Sanncrud et al., 1989) and changes in GABA functioning (Gonsalves and Gallager, 1985, 1987; L&her and Stephens. 1988; Tietz et al., 1989). In addition, chronic exposure to benzodiazepine agonists have also been reported to alter the effects of benzodiazepine antagonists and

1OS7: Ttiksda t‘t al., 19S9). For example, previously behaviorally inactive hcnzodiazepine receptor antagonists prl~ke a nithdrawal syndrome (including signs of vomiting. abnormal postures and seizures) in ~~~~-~urn~n primates after a period of chronic agonist admin~str~~tion (Cumin et al.. 19X2; Lukas and Griffit&. 1982, 1085: Lamb and Griffiths, 1984). Studies in mice have shown an increased sensitivity to both the effects of antagonists and inverse agonists after chronic agonist treatments. suggesting a shift in efficacy (termed ‘withdrawal shift’) along a continuum of activity in the direction of the inverse agonist (Little et al.. 1987). Benzodiazepine receptor ligands can be useful tools tur analyzing the bidirectional effects mediated at the GABA/benzodiazepine receptor complex and can be used to help elucidate the mechanisms of receptor alterations that occur during chronic agonist administration. The present study was undertaken to investigate the Behavioral effects of a benzodiazepine agonist, an antagonist. and two inverse agonists in non-human

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ATAXIA

primates using observational methods previously shown sensitive to the effects of these compounds. One set of experiments characterized the effects of acute administration of several benzodiazepine ligands alone and in combination in non-dependent baboons. The compounds investigated were the agonist midazolam, the antagonist flumazcnil. the inverse agonists FG7142 and $carboethoxy-P-carboline hydrochloride (PCCE); intcractions among midazolam, flumazenil and pCCE were also studied. A second set of experiments assessed the effects of acute administration of most of these same benzodiazepine ligands, alone and in combination. in diazepam-dependent baboons. More specifically, several experiments characterized the effects of the agonist midazolam, the antagonist flumazenil. the inverse agonist pCCE and the interactions of midazolam or flumazenil in combination with PCCE in diazepam dependent baboons. Other experiments characterized the effects of acate ;I.dministration of flumazenil and PCCE in baboons withdrawn from diazepam.

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wW Wm.) Fig. 1. Behavioral effects produced by midazolam. flumuzenil. FG7142 and BCCE iri the non-dependent condition. Behavioral signs were scored ContinuwslY during a I-h observational session after i.m. administration of vehicle or drug. X axes: drug dose, log scale; vehicle (V). Y axes: the number of min during the M-min observational session in which the signs appeared. Dana points show means (n = 3-S). Brackets indicate I S.E.; upper or lower bracket hns somelimcx heen deleted for clarity.

161

2. Materials and methods 2.1. Subjects Eight male baboons (Papio cynocephalus of anubis and cynocephalus types; Primate Imports, NY, NY) weighing 19-37 kg were used as subjects in one or more of the three experimental conditions: non-dependent condition, dependent condition or withdrawn condition. Non-dependent baboo: s (BE, MI, TH, OP, AR, OM) were not catheterized. Dependent baboons (CH, NU, OM, AR, BE, MI, TH) and withdrawn baboons (CH, NU, BE, MI, TH) were surgically implanted with intragastric (i.g.1 catheters (Lukas et al., 1982) and maintained on 20 mg/kg per day i.g. diazepam. Immediately before the experiment, these baboons had been continuously maintained on diazepam for 3 months to 1 year. These baboons were tolerant to the behavioral effects of diazepam; there were no behavioral signs of sedation prior to the start of these conditions. In the withdrawn condition, vehicle was substituted for diazepam for 4 days prior to testing.

3O-

For those baboons which served as subjects in multiple conditions, the sequence of conditions varied across subjects. Baboons BE, MI, TH served as subjects sequentially in the non-dependent condition, dependent condition, and withdrawn condition. Baboons AR and OM completed the dependent condition and, after 4 months of being drug free, were tested in the non-dependent condition. Baboons CH and NU completed the dependent condition and then were tested in the withdrawn condition. Baboons had continuous ad libitum access to water via a drinking tube and to monkey biscuits (Purina Monkey Chow or Charles River Biscuits). Baboons received two pieces of fresh produce and a multivitamin daily. 2.2. Apparatus Baboons were housed in standard stainless steel primate cages with a bench running the length of one wall. Each cage, which served as the experimental chamber, was surrounded by a sound attenuating dou-

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in combination with 0.32 mg/kg midazolam (n = 4) in non-dependent effects of /3CCE nlone (n = _5) and fiCCE Dose-effect functions for fiCCE alone uru redrnwn from fig. I. Details as in fig. I.

baboons.

conducted by pairs of indcpcndent nerud ct al., 1YXY).

ed ply~vood estern&tl enclosure and was continuously illuminated with a 20-W frosted bulb.

The

behavioral

effcccts of benzodiazcpinc

(San-,

Acute dose-response functions for the bcnzodiazepinc agonist midazolam. the antagonist flumazenil and two inverse agonists, FG7142 and pCCE were generated in non-dependent baboons. Although both FG7142 and pCCE arc often described as inverse agonists, there are a variety of biochemical and phar,macological differences between these compounds (Petersen et al., 1982, 1983). A single dose of test drug was administered immediately before each observational session: mida?cXlam (0.01-3.2 mg/kg i.m.; baboons BE, MI. TH, AR), flumazenil (0.1-32 mg/kg im.; baboons BE, MI, TH), FG7142 (0.1-100 mg/kg i.m.; baboons Ml, TH, OP) and PCCE (0.1-32 mg/kg, i.m.; baboons BE, MI, TH, AR, OM). Seven to ten days elapsed between injections of individual drug doses. Interactions among thy benzodiazepine agonist midazolam, the antagonist flumazenil, and the inverse agonist pCCE were also studied in non-dependent

rcccptor ligands \vcre assessed in baboons using the behavioral rating scale previously described by Sannerud et al. (1%9X On test days. an acute dose of drug was administcrcd to an individual baboon immediately before a 1-b observational session. The observational session permitted the scoring of a wide variety of behaviors, postures. movements and position in cage. and the assessment of coordination, level of activity. sedation and tremor. During the l-h observational session, bcha;ior was recorded continuously, and the passage of time was recorded at I-m;n intrrva!s. The presence or absence of signs in each I-min block was tabulated. Thus, the maximum score for each sign in a I-h obscrvation period was 60. Observations were performed by observers blind to the drug conditions on 35% of the sessions. Previous studies using these procedures showed a highly significant degree of reliability behveen the overall scores from observational sessions

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in combination

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condition.

baboons to assess the ability of the benzodiazepine Iigands to mutually antagonize their behavioral effects, Three studies were conducted. First, a single dose of flumazenil was administered in combination with a range of /3CCE doses: Rumazenil(3.2 mg/kg i.m.1 and pCCE (0.1, 1.0, 10 mg/kg i.m.1 (baboons BE, MI, TH, AR, OMl. Second, a single dose of midazolam was administered in combination with a range of pCCE doses: midazolam (0.32 mg/kg i.m.) and PCCE (0.1, 1.0, 10 mg/kg i.m.1 (baboons BE, MI, TH, AR). Third, a single dose of Gdazolam was administered in combination with a single dose of flumazenii: midazolam (3.2 mg/kg i.m.1 and flumazenil 13.2 mg/kg i.m.1 (baboons TM, AR, OM).

fourteen days elapsed between injectiors of individual drug doses. Combinations of the benzodiazepine antagonist flumazenil and the inverse agonist PCCE were also administered to dependent baboons in order to assess the ability of flumazenil to antagonize or potentiate the behavioral effects of PCCE. Two doses of flumazenil were administered in combination with a range of pCCE doses: flumazenil (3.2, 10 mg/kg i.m.1 am’ PCCE (0.32, 3.2, 32 mg/kg im.1 (baboons CH, NU. OM).

Acute dose-response functions for flumazenil and $lCCE were generated in baboons during diazepam withdrawal. In this experiment, diazepam-dependrnt baboons were administered vehicle via continuous i.g. infusion for 4 days prior to each dose of ffumazenil or JXCE. The baboons were returned to the chronic 20 mg/kg per day Lg. diazepam condition for two weeks after each dose of ~umazenil or PCCE. Each dose of flumazenil or PCCE was administered immediately before an observational session: flumazenil (0.032 10 mg/kg Lm.; baboons BE, MI, TH, CH, NW, ji?CCE (0.032-5.4 mg/kg i.m.; baboons BE, MI. TH, CH). Eighteen days elapsed between injections of individua!

Acute dose-response functions for the benzodiazepine agonist midazolam, the antagonist fl umazenil and the inverse agonist pCCE were generated in benzodiazepine dependent baboons maintained on 20 mg/kg per day diazepam via continuous Lg. infusion. A single dose of test drug was administered immediately before an observational session: midazolam (0.32100 mg/kg Lm.; baboons CX, NW, flumazenil (1.0-100 mg/kg i-m.; baboons CH, NU, OM) and PCCE (0.321Oi.lmg/kg i.m.; baboons CH, NU, OM, AR). Ten to

307

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Fig. 6. The behavioral effects of PCCE in the non-dependent (n = 5). diazepam-dependeiit (n = 4) and withdrawn (n = 4) conditions. X axes: dose of PCCE. log scale. Y axes: percentage of baboons (left panel) or the number of min during the 60-min observational session in which the signs appeared (right panel). Other details as in fig. 5.

Midazolam, the benzodiazepine agonist, produced dose-related increases in signs indicating sedation: ataxia (fig. l), lip droop, falls and sedated postures (i.e. sitting or laying down with eyes closed) (fig. 4, non-dependent condition). None of these effects were produced by midazolam vehicle or the other drugs tested. Flumazenil, the benzodiazepine receptor antagonist, was behaviorally inactive: it did not produce a meaningful change in any of the behavioral signs across the wide range of doses evaluated. Figure 1 illustrates the lack of behavioral effect of flumazenil across six classes of signs that were sensitive to one or more of the other drugs tested. The inverse agonists, FG7142 and PCCE, produced unique profiles of behavioral effects that were different than those observed after midazolam and flumazenil administration. The effects produced by FG7142 and pCCE were similar in that both produced dose-related increases in socially withdrawn posture and limb tremor (fig. 1). The compounds were different in that FG7142 produced dose-related increases in nose rubbing (nnt shown), while pCCE increased vomiting and several proconvulsant behaviors (twitch/jerks: isolated spasms of head or limbs, and myoclonic jerks: symmetrical clonic spasms of bilateral limbs usually preceding seizures). In addition, pCCE (32 mg/kg) produced seizures (major convulsion or tonic spasms followed by clonic jerking and prolonged depression) in three of the five baboons (fig. 6, non-dependent condition).

Figure 2 shows the dose-response functions for PCCE alone and PCCE in combination with a behaviorally active dose of midazolam. As shown, 0.32 mg/kg midazolam produced an increase in ataxia when administered with pCCE vehicle; /?CCE produced dosedependent antagonism of this effect. The figure also shows that the effects produced by 10 mg/kg PCCE alone (socially withdrawn posture, limb tremor, vomiting, twitch/jerks and myoclonic jerks) were completely antagonized by midazolam. Figure 3 shows the dose-response functions for PCCE alone and PCCE in combination with a dose of flumazenil. Consistent with the results shown in fig. 1, 3.2 mg/kg flumazenil administered with PCCE vehicle did not alter any of the behavioral signs evaluated. Figure 3 shows that the behavioral signs produced by IO mg/kg pCCE alone (body tremor, socially withdrawn posture, limb tremor, vomiting, twitch/jerk and myoclonic jerks) were completely antagonized by flumazenil. Not shown graphically, flumazenilf3.2 mg/kg) completely antagonized the the sedative signs produced by midazolam (3.2 mg/kg): ataxia, lip droop, falls and sedated postures (e.g. sitting or laying down with eyes closed).

3.2. Drug interactions in the non-dependent condition

There were quantitative and qualitative differences among the dose-effect functions generated for the benzodiazepine ligands in the three conditions of this study. Figure 4 shows that midazolam was virtually without effect in dependent baboons, in contrast to the dose-

Assessment of the interactions between midazolam, flumazenil and PCCE showed that each ligand could antagonize the behavioral effects of each of the other ligands.

3.3. Cotnparisop of dose-effect fmctions in non-dependent, diazepam dependent and withdrawn conditions

dependent increases in ataxia, lip droop. falls and sedated postures observed in non-dependent baboons. Figure 5 shows that in contrast to flumazenil’s lack of effects in the non-dependent condition, in dependent and withdrawn conditions, flumazenil produced dose-related increases in signs previously shown sensitive to the inverse agonist. Not shown in the fig.. in no condition did flumazenil affect signs previously shown sensitive to midazolam fataxia. lip droop, falls. sedated postures). In addition, flumazenil was substantially more potent in its ability to produce these effects in the withdrawn than in the dependent condition: there were 3.2- to IOO-fold differences in the peak effects for socially withdrawn posture, limb tremor, vomiting, twitch/jerks and myoclonic jerks in withdrawn baboons compared to dependent baboons. pCCE tended to be more potent in producing seizures in the withdrawn condition than in the nondependent and dependent conditions (fig. 6, left panel). For example, @CCE (IO mg/kg) produced tonic-clonic seizures in all four of the withdrawn baboons, but produced seizures in only one of the five non-dependent baboons, and in none of the four diazepam-dependent baboons. The increased frequency of seizure in the withdrawn condition modified the expression of other behavioral signs during the observational sessions. The PCCE-produced convulsions, which typically occurred 15-20 min after injection, were followed by a prolonged period of post-ictal behavioral depression. which is reflected in the dose-related increases in socially withdrawn postures (fig. 6, right panel). 3.1. Drug interactions in the diazepam-dependent tion

condi-

Flumazenil administered in combination with PCCE in the diazepam-dependent condition did not consistently potentiate or antagonize PCCE’s behavioral effects. Compared with PCCE alone, flumazenil (3.2 and 10 mg/kg) in combination with PCCE (0.32, 3.2 and 32.0 mg/kg) produced alterations in the frequency of withdrawal signs, such as vomiting, myoclonic jerks, and seizures, but these effects did not appear to be dose-dependent (data not shown).

4. Discussion The present study found that the behavioral effects of benzodiazepine agonists, antagonists, and inverse agonists can be differentiated using a behavioral rating scale, and that the behavioral effects observed in the non-dependent condition can be modified by chronic diazepam administration in the dependent and withdrawn conditions. In the non-dependent condition, the behavioral effects of benzodiazepine receptor ligands

were dose-dependent and appeared to lie on the continuum of behavioral activity ranging from benzodiazepine agonist to benzodiazepine inverse agonist effects: midazolam produced sedation and ataxia, fhtmazenil prodiiced no behavioral effects, FG7142 produced limb tremor, and PCCE produced twitch/ jerks, vomiting, myoclonic jerks and seizures. However, in the diazepam-dependent and withdrawn conditions, the spectrum of behavioral effects produced by benzodiazepine receptor ligands appeared shifted in the direction of the inverse agonist: midazolam produced no effect in the dependent condition, flumazenil produced limb tremor, twitch/jerks, vomiting, and socially withdrawn postures in both dependent ard withdrawn conditions, and pCCE was more potent at producing seizures in the withdrawn than in the non-dependent condition. The present study behaviorally differentiated between FG7142 and pCCE on the basis of their convulsant effects; FG7142 produced only limb tremor, and PCCE produced twitch/ jerks, myoclonic jerks and seizures. Although both compounds are often labelled as inverse agonists and have been shown to produce proconvulsant effects under some conditions (ThiGbot, et al., 19881, the present results are consistent with preclinical research using an electroshock seizure model in mice that demonstrated that, in contrast to PCCE, FG7142 produces anticonvulsant effects at high doses (Petersen et al., 1983). It is possible that the absence of convulsant effects after FG7142 noted in the present study reflects the lower intrinsic activity at the benzodiazepine receptor complex of FG7142 compared to PCCE. The production of differential behavioral effects by benzodiazepine receptor ligands in baboons is consistent with the 3-state receptor model of the GABA/ benzodiazepine receptor complex; the wide range of behavioral effects produced by benzodiazepine receptor ligands can be related to their bidirectional modulatory effect on the inhibitory neurotransmitter GABA (Pole et al., 1982; Haefely, 1990; Little et al., 1987) . Acute administration of benzodiazepine agonists increase GABA functioning and produce ataxia and sedation in animals and humans (cf. Richards et al., 1986; Haefely, 1988; Woods et al., 19871. The benzodiazepine antagonist, flumazenil, has virtually no intrinsic effects on GABA in animals or humans at doses that antagonize benzodiazepine agonists (cf. Klotz and Kanto, 1988), although flumazenil has been reported to produce weak agonist or inverse agonist effects when administered in high doses (cf. File and Hitchcott, 1990). The inverse agonists FG7142 and pCCE are reported to decrease GABA functioning, and produce behavioral agitation in animals and ‘anxiogenic’, effects in humans (cf. Nutt, 1983; Dorow et al., 1987; ThiCbot et al., 1988).

167

In the present study, the behavioral effects produced by the benzodiazepine ligands in the non-dependent condition appeared to be receptor-mediated; administration of combinations of receptor figands (midazolhm + PCCE, midazolam i- flumazenil, or flumazenil -t PCCE) showed that each ligand could antagonize the effects of the other ligands. In the con-dependent condition, flumazenil antagonized the effects of both midazolam and PCCE, and co-administration of midazolam and /3CCE produced a mutual antagonism of their behavioral effects. Consistent with the results in the present study, other investigators found that the interactions of benzodiazepine receptor agonists, antagonists and inverse agonists resulted in antagonism or mutual antagonism of many of their classic behavioral effects, as well as their discriminative stimulus effects (Oakley and Jones, 1980; Tenen and Hirsh, 1980; File and Baldwin, 1987; Ongini et al., 1983; Shannon et al., 1388; Valin et al., 1982; Schweri et al., 1982; File and Pellow, 1984; Dorow et al., 1983; Takada et al., 1986; Barrett et al., 1985; Wettstein 1989). Relative to the non-dependent condition, chronic diazepam administration altered the behavioral effects of the receptor ligands: (1) mid~olam administration produced no behavioral effect in the dependent condition (i.e. tolerance developed to the sedative effects of midazolam which were observed in the non-depenllent condition); (2) in contrast to flumazenil’s lack of behavioral effects in the non-dependent condhion, flumarenil administration in the diazepam-dependent and withdrawn conditions precipitated a withdrawal syn. drome that consisted of an inverse agonies-like profile of behavioral effects: (3) PCCE produced seizures in the withdrawn condition at doses that were substantially lower than those required to produce seizures in the non-dependent and dependent conditions; (4) in contrast to ~umazenil’s antagonism of j%XE’s behavioral effects in the non-dependent condition, ffumazcnit given in combination with @CCE in the depcndcnt condition produced changes in the frequency of withdrawal signs, but did not consistently potentiate or antagonize the effects of PCCE. These alterations in behavioral effects confirm an3 extend previous research suggesting that chronic administration of benzodiazcpine agonists produce changes in the behavioral effects of the receptor fig ands, with the effects shifting afong a continuum of activity toward the direction of the inverse agonist (Little et at,, 1986; Nutt and Costello, 1988) . This phenomenon, which has been termed ‘withdrawal shift’. can be observed as a precipitated withdrawal syndrome after administration of an intrinsically neutral receptor antagonist or as potentiation of the behaviorai effects of inverse agonists. Flumazenii, for example, had no effect on behavior when given afonc, but produced a precipitated withdrawal syndrome during chronic bcn-

zodiazepine agonist administration in rats, cats, dogs and baboons (Boisse et al., 1986, 1990, Wilson and Gallager, 1988; Giorgi et aI., 1989; Lascher et al., 1989; McNichols et al., 1988; Lukas and Griffiths, 1982, 19134: Lamb and Griffiths 1984; Sannerud et at., 198929).In addition, three benzodiazepine antagonists (CGS 9895, P-CCtB, ~umazenif) that had no effect on schedufecontrolled behavior, produced disruptions of this behavior during chronic administration of chlordiazepoxide in rats (Talcada et al., 198% . An analogous shift in potency of inverse agonists after termination of flurazepam or Iorazepam treatment was observed as sensitization to the convulsant effects in rodent (Little et al., 1987, Nutt and Costello, 1988; Petersen and Jensen 1987a, b; Schatzki et al., 1989). Taken together, these data suggest that chronic administration of benzodiazepine agonists induce an increase in efficacy or potency of the benzodiazepine receptor figands to produce inverse agonist-like effects. In the dependent condition of the present study, the insensitivity to PCCE and flumazenil may be explained by a competitive interaction of diazepam with these drugs at the receptor. It is probable that chronic infusion of diazepam produced an accumufation of high concentrations of diazepam and its active metabolites. It seems likely that the presence of those receptor agonists in the dependent condition could have resuited in rightward shifts in the dose-response functions for ~umazenil and #?CCE compared to the withdrawn condition. These data are consistent with previous reports of a lack of a precipitated withdrawal syndrome by ~-carbofine derivatives FG 7142 and ZK 93426 in diazcpam-dependent cats and the suggestion that this inability may be due to pharmacokinetic factors, such as the presence of high brain lcvcls of diazcpam or its metabolites at the receptor (Ongini et at., 1985; Giorgi et at., 1988). Chronic diazepam administration has been shown to produce a number of biochcmic:rl alterations that could possibly influence the expression of the behavio~;~l effects of benzodiazepinc receptor ligands, The change in behavioraf efficacy of ~c~odiazepinc receptor figands during chronic diazepam administration may be related to perturbation in the GABA/ bcnzodiazepinechloride receptor complex, includil~g receptor downregulation, long-term decreases in GABA sensitivity, changes in GABA/bcnzodiazepine c(~upling and/or decreases in GABA efficacy (C&salves and CUlagcr, 1985; Gaffager et al., 1985; Wcninger and Gallager, 1988: Tietz et al., 1989). In addition. chronic diazcpam administration has been reported to increase the production of a putative endogenous inverse agonist-like ligand, diazepam binding inhibitor (DBI) and its precursors (Miyata ct al., 1987). Although biochemical changes during cfuonic diazcpam administration are likely to undcrlic the dccrcascd cffcctivcncss of benzo-

diszepine

agonists (i.e.

tolerance).

tate

and the incrcascd

abstinence

Biochern.

efficicacg or potency of flumazenil and /3CCE to produce convulsant effects (sensitization) in the depcn-

Gonsalves.

dent a!d withdrawn conditions, the precise mechanism(s) responsible for these behavioral alterations are unknown and warrant further study.

Gonsalves.

signs in diazepam-dependent

S.F.

and

IS-178%induced

D.W.

Gallager.

reversal

Gallager.

after chronic diazepam.

Brain Res. 405. 94.

W..

1990. The

thank

S. Womack.

supported During

S. Knipp.

E. Koehler

assistance in conductiug

by National

Institute

on Drug

this project. C.A. Sannerud

tute on Drug Abuse National

and S. James for

this study. This

study was

Abuse Gr..tnt DA

01 147.

was supported hy National

Research

Service Award

DA

Insti-

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