PET analysis of alcohol interaction with the brain disposition of [11C]flumazenil

Acute alcohol administration to rats has in preliminary studies been reported to drastically increase the binding of the benzodiazepine (BZ) receptor antagonist [³H]flumazenil (Ro 15-1788) to central BZ receptors. In the present study the effect of acute alcohol ingestion on the disposition of [¹¹C]flumazenil in the human brain and plasma was examined by positron emission tomography (PET) in four healthy volunteers. Neocortex, cerebellum and pons (reference region) were delineated using X-ray computerized tomography (CT). Alcohol did not increase either total radioactivity uptake or specific [¹¹C]flumazenil binding in neocortex or cerebellum. However, alcohol had a small but significant effect on [¹¹C]flumazenil in arterial blood. After alcohol the plasma radioactivity peak was higher, more narrow and occurred earlier than in the control experiments. The present experiments contradict the view that alcohol directly affects central BZ receptor binding in man. Thus the dramatic increase of flumazenil binding in rat brain reported previously could not be observed in the human brain.     

Chronic clonazepam administration induced benzodiazepine receptor subsensitivity

Clonazepam and chlordiazepoxide were administered chronically in increasing doses for three weeks in two different strains of mice. Forebrain [³H]diazepam binding was assayed in groups of mice sacrified at 2, 26, 50 hr and 10 days following the last dose. Scatchard and single point analyses revealed a significant decrease in the number of [³H]diazepam binding sites [B_max] which persisted for at least two days following chronic clonazepam treatment. The B_max changes observed following chlordiazepoxide treatment were less pronounced than those elicited by clonazepam. No significant changes in receptor binding affinity (K_d) were detected with either drug. In the clonazepam-treated animals, B_max values returned to normal by day 10 after drug treatment. Chronic benzodiazepine administration therefore induced a decrease in the apparent number of benzodiazepine binding sites in the mouse forebrain. The magnitude and duration of the observed subsensitivity appears to depend on the potency of the administered benzodiazepine.     

A PET study of D1-like dopamine receptor ligand binding

Rationale: Several positron emission tomography (PET) studies have shown that radioligand binding to D₂-like dopamine receptors competes with endogenous dopamine. Objective: The purpose of this PET study was to examine the effect of amphetamine and reserpine on D₁-like dopamine receptor binding. Methods: Three Cynomolgus monkeys were examined with the radioligands [¹¹C]SCH 23390 or [¹¹C]NNC 112 at baseline condition and after pretreatment with amphetamine (2 mg/kg IV). The B/F values (binding potential) in the striatum and the neocortex were calculated at transient equilibrium. In two monkeys, the effect of the long-lasting dopamine depletion after reserpine (1 mg/kg IV) was followed by a repeated Scatchard procedure in up to 77 days after drug administration. The Scatchard analysis was based on two PET measurements with high and low specific radioactivity and allowed the calculation of D₁-like dopamine receptor density (B_max) and apparent affinity (K_D ^app). Results: The effect of amphetamine on the B/F values was between –14 and 6%. These changes can be considered as within the range of the test-retest reliability. Thus, there was no evident effect of amphetamine-induced dopamine release on D₁-like dopamine receptor binding. Five hours after reserpine administration, there was no change in B_max or K_D ^app. At 3, 23, and 28 days after reserpine administration, the Scatchard analyses indicated a 13–20% reduction in B_max without any evident change in K_D ^app in both the striatum and the neocortex. Conclusions: The lack of evident effects of amphetamine and reserpine on D₁-like dopamine receptor binding is markedly different from the 20% amphetamine-induced decrease and 50% reserpine-induced increase that has been consistently reported for D₂-like dopamine receptor binding. The data indicated that D₁-like dopamine receptor occupancy of endogenous dopamine is low at physiological condition. It is thus unlikely that D₁-like dopamine receptor radioligands can be used to measure changes in the concentration of endogenous dopamine. Received: 8 March 1999 / Final version: 12 May 1999     

In vivo binding of N-n-propylnorapomorphine in the rat striatum: Quantification after lesions produced by kainate, 6-hydroxydopamine and decortication

The neuronal localization of in vivo N-n-propylnorapomorphine (NPA) binding in the rat striatum was studied using 3 types of lesions. Striatal dopamine (DA) receptor densities (B_max) were estimated from the relationships between total striatal and cerebellar NPA accumulation. A B_max of 26.9 ± 1.6 fmol · mg^?1 wet weight tissue was found in the striata of non-lesioned (unoperated) rats. Similar values were obtained for striata with 6-hydroxydopamine-lesioned dopaminergic fibres. Kainate (KA)-lesioned striata contained 4.6 ± 0.5 fmol · mg^?1 saturable NPA binding sites. After unilateral decortication the receptor densities were altered in both striata resulting in ipsi- and contralateral B_max values of 23 and 36 fmol · mg^?1 respectively. With a tracer dose of [³H]NPA less radioactivity accumulated in the KA-lesioned striatum, while after unilateral destruction of the dompaminergic pathway more radioactivity was found in the ipsilateral striatum and no bilateral differences in striatal radioactivity concentration were found after unilateral cortical ablation. These observations show that all in vivo saturable striatal NPA binding sites are situated on striatal neurons and cortico-striatal afferents and that the effects of lesions on striatal DA receptor densities cannot be predicted from bilateral differences in the accumulation of tracer doses of [³H]NPA.     

Effect of ACTH,adrenalectomy and the combination treatment on the density of 5-HT2 receptor binding sites in neocortex of rat forebrain and 5-HT2 receptor-mediated wet-dog shake behaviors

The effect of ACTH and/or adrenalectomy on serotonin (5-HT)₂ receptor binding sites was evaluated in the neocortex of rat forebrain. One day after the adrenalectomy or sham operation, ACTH (50 µg/day) was injected subcutaneously into adult male SD rats for 10 consecutive days. Saturation analysis showed that subchronic ACTH treatment significantly increased the B_max values for³H-ketanserin binding without any change in the K_d values. Moreover, this ACTH-induced increase in the B_max values was prevented by adrenalectomy. The concentrations of 5-HT and 5-hydroxyindole acetic acid (5-HIAA) measured by HPLC-ECD were not altered by these manipulations. Ten-day administration of corticosterone (20 and 50 mg/kg) also increased 5-HT₂ receptor density in the neocortex of rat forebrain. 5-HT₂ (and 5-HT_1C) receptor agonist, (±)DOI-induced wet-dog shakes in ACTH and/or adrenalectomy-treated rats were also examined. Ten-day administration of ACTH enhanced (±)DOI-induced wet-dog shakes and this increase was prevented by adrenalectomy. These results indicate that subchronic adrenocorticotropinadrenal axis activation of rats increases both the number of 5-HT₂ receptors in neocortex of forebrain and the wet-dog shake responses induced by (±)DOI.     

[11C]Ro 15-4513, a ligand for visualization of benzodiazepine receptor binding

Ro 15-4513, a partial inverse agonist at the benzodiazepine (BZ) receptor site was labelled with¹¹C and used for in vitro autoradiography on human post mortem brain sections and for positron emission tomography (PET) on Cynomolgus monkeys. The total radiochemical yield of [¹¹C]Ro 15-4513 was 30–40% with an overall synthesis time of 40 min. The specific radioactivity was about 1000 Ci/mmol at end of synthesis. In vitro autoradiography showed that [¹¹C]Ro 15-4513 bound specifically predominately in the neocortex of the human brain. Specific binding was also demonstrated in the basal ganglia and the cerebellar cortex. Flumazenil (Ro 15-1788) and clonazepam inhibited the binding in cerebral regions, but a significant proportion in the cerebellum was not inhibited by these agents. This proportion may represent ₆-containing BZ receptors. PET examination of [¹¹C]Ro 15-4513 binding in Cynomolgus monkeys demonstrated high uptake of radioactivity in neocortex. The uptake of radioactivity was markedly displaced by high doses of Ro 15-4513 or clonazepam. [¹¹C]Ro 15-4513 should be a useful ligand to examine BZ receptor characteristics in the living human brain by PET.     

Flunitrazepam photoaffinity labeling of the GABAA receptor reduces inhibition of [3H]Ro15-4513 binding by GABA

The benzodiazepine drugs modulate γ-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA_A receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [³H]Ro15-4513, [³H]flumazenil (Ro15-1788), and [³H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [³H]flunitrazepam and [³H]flumazenil, but increased the binding of [³H]Ro15-4513. The increase in [³H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent K_d, with no change in B_max. Following photoaffinity labeling, negative modulation of [³H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [³H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [³H]Ro15-4513.     

Central benzodiazepine receptor autoradiography in hippocampal sclerosis

1. The γ-aminobutyric acid (GABA)_A/central benzodiazepine receptor (cBZR) complex is a major inhibitory receptor in the vertebrate CNS. Binding of [¹¹C]-flumazenil to this complex in vivo is reduced in hippocampal sclerosis (HS). It has been uncertain whether reduced cBZR binding is entirely due to neuronal loss in HS.
2. The objective of this study was to characterize abnormalities of the cBZR in HS with a correlative autoradiographic and quantitative neuropathological study.
3. Saturation autoradiographic studies were performed with [^†H]-flumazenil to investigate relationships between neuronal density and receptor availability (B_max) and affinity (K_d) in HS. Hippocampal tissue was obtained at surgery from 8 patients with intractable temporal lobe epilepsy (TLE) due to HS and autopsies of 6 neurologically normal controls. Neuronal densities were obtained by means of a 3-D counting method.
4. B_max values for [^†H]-flumazenil binding in the subiculum, CA1, CA2, CA3, hilus and dentate gyrus were all found to be significantly reduced in HS compared with controls and significant increases in affinity were observed in the subiculum, hilus and dentate gyrus. In HS, cBZR density in the CA1 region was significantly reduced (P<0.05) to a greater extent than could be attributable to neurone loss. In other regions, B_max was reduced in parallel with neuronal density.
5. In HS, there is a loss of cBZR in CA1 over and above loss of neurones. This finding and increases in affinity for flumazenil in subiculum, hilus and dentate gyrus imply a functional abnormality of the GABA_A/cBZR complex that may have a role in the pathophysiology of epileptogenicity in HS.

     

Benzodiazepine receptor increase following repeated pentylenetetrazole injections

Daily injections of a subconvulsant dose of pentylenetetrazole (PTZ) caused a mild ‘kindling’ response and a significant increase in forebrain benzodiazepine receptor B_max. No changes in affinity for [³H]diazepam or PTZ in vitro were observed. Mice given equal numbers of injections on a 3/week schedule developed significant ‘kindling’, increased forebrain receptor B_max and decreased PTZ affinity. B_max changes were uncorrelated with ‘kindling’ response and may result from PTZ acting directly as an antagonist at benzodiazepine receptors.     

Effect of fluvoxamine on platelet 5-HT2A receptors as studied by [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy volunteers

Alterations in platelet 5-HT_2A receptor characteristics have been reported in major depression as well as in other psychiatric diseases, and some effort has been made to utilize platelet 5-HT_2A receptor status as a biological correlate to antidepressant drug response. In order to investigate whether treatment with a selective serotonin reuptake inhibitor affects platelet 5-HT_2A receptors, we have studied platelet [³H]lysergic acid diethylamide ([³H]LSD) binding in healthy subjects treated with fluvoxamine in increasing dosage once weekly for 4 weeks. After 1 week of fluvoxamine treatment (25 mg/day), both B_max and K_d were significantly lower than before the start of the treatment (19.9 versus 25.5 fmol/mg protein, P = 0.005 for B_max; 0.45 versus 0.93 nM, P = 0.006 for K_d). B_max returned to baseline during week 2, whereas K_d was lower than the baseline value throughout the treatment period. After discontinuation of fluvoxamine treatment, there was a significant increase in K_d (0.50 nM before discontinuation vs. 1.14 nM after discontinuation; P = 0.001), but not in B_max. The study demonstrates that fluvoxamine affects platelet 5-HT_2A receptor status irrespective of underlying psychiatric disease, and that this effect is evident already after 1 week at a subtherapeutic fluvoxamine dose. Received: 11 October 1996/Final version: 12 March 1997     

A PET Study on the Acute Effect of Ethanol on Striatal D2 Dopamine Receptors with [11C]Raclopride in Healthy Males

The effect of acute oral ethanol intake (1·0 g/kg) on cerebral D2-receptors ([¹¹C]raclopride binding) was studied in seven healthy volunteers, using water and alcohol in two separate 59-min PET sessions. In the alcohol experiments, the blood ethanol concentration at the beginning of the imaging was 26·4±3·8 mmol/l and remained stable during the PET session. Ethanol was not found to influence binding of [¹¹C]raclopride to the dopamine D2 receptors in the human striatum, as indicated by the unchanged ratio B_max/K_d of the whole (left and right) striatum. The T_max values of the control and ethanol experiments did not differ in the whole striatum, but the right to left difference of striatal T_max was turned from negative to positive by ethanol (P=0·02). However, the difference between hemispheres in B_max/K_d was not significantly altered by ethanol intake. There was considerable interindividual variation in the response of all the above parameters to acute ethanol exposure. According to the present results, the acute effects of peroral ethanol exposure on striatal D2 receptor binding potential are of relatively small magnitude in man. However, the changes in T_max suggest that ethanol may influence the right–left difference of [¹¹C]raclopride binding. © 1997 by John Wiley & Sons, Ltd.     

Striatal D2 dopamine receptor binding characteristics in vivo in patients with alcohol dependence

Striatal D₂ dopamine receptor characteristics of nine male patients with alcohol dependence abstinent for 1–68 weeks and eight healthy male volunteers were studied in vivo with positron emission tomography. The selective D₂ receptor ligand [¹¹C]raclopride and equilibrium model was used for D₂ receptor density (B_max) and affinity (K_d) measurements. A trend for a decreased striatal D₂ receptor density and for reduced D₂ receptor affinity was observed in patients with alcohol dependence. These parameters were not statistically significantly different between alcoholics and controls, but the ratio between D₂ receptor density and affinity (B_max/K_d or the striatum/cerebellum ratio from the high specific activity scan) was highly significantly lower in alcoholics than that of controls. In conclusion, the low D₂ dopamine receptor B_max/K_d ratio (striatum/cerebellum ratio) indicates that specific aspects of striatal [¹¹C]raclopride binding in vivo are deviant in alcoholics compared to controls. The result is compatible with a reduced avidity of striatal dopamine D₂ receptors in alcoholics, which is in line with the idea that D₂ dopaminergic mechanisms are involved in the biology of alcohol dependence in man.     

Graphical orientation procedure for the estimation of K d and B max values of ligand binding to two receptor subpopulations

Summary Within a few minutes, the procedure described here provides to the investigator, performing binding studies, information about K _d and B _max values of two possibly present receptor subpopulations. The procedure is based on the comparison of the experimental curve with values of ligand binding to only one receptor population at 4 defined levels of radioligand binding. The binding parameters are derived from comparison of 4 horizontal deviations at these levels with tabulated deviations. K _d and B _max values can be further confirmed by comparison of the experimental data with the simulated binding curve obtained by entering the binding parameters found in the formula for ligand binding to two receptor subpopulations. The practical use of this procedure was demonstrated both for simulated and for experimental data; it was confirmed by comparison with binding parameters obtained by computer programs used for the evaluation of receptor heterogeneity.     

Studies on the binding of [3H]flumazenil and [3H]sarmazenil in post-mortem human brain

Two benzodiazepine analogues, [³H]flumazenil and [³H]sarmazenil, were used to study the GABA_A/benzodiazepine receptor complex in human post-mortem brain using in vitro receptor assays on homogenates and whole hemisphere autoradiography. Both radioligands bound in a saturable manner to single binding sites in the tissue preparations from any brain region. The highest levels of binding were found in the cortical regions and in cortex cerebelli. Both [³H]flumazenil and [³H]sarmazenil were excellent radioligands for autoradiography with high binding in cerebral and cerebellar cortex with no or very low binding in areas with white matter. The addition of a high concentration of flumazenil or clonazepam did not inhibit the binding of [³H]sarmazenil to granule cells in the cerebellum while the binding of [³H]flumazenil was abolished completely in all regions. The results show that with the two different radioligands, one an antagonist and one a partial inverse agonist, the binding pattern to GABA_A/benzodiazepine receptor complex is approximately similar in most brain regions. The additional binding seen in the cerebellum with [³H]sarmazenil is suggested to be due to binding to an α₆-containing complex.     

Cerebral cortical 5-HT1, and 5-HT2, receptors of morphine tolerant-dependent rats

The effect of chronic administration of morphine to rats on 5-HT₁ and 5-HT₂ receptors in the cerebral cortex was determined. Male Sprague-Dawley rats were implanted subcutaneously with 6 pellets of morphine (each containing 75 mg of morphine free base) during a 7 day period. Animals which served as controls were implanted with placebo pellets. The procedure for implantation of pellets produced a high degree of tolerance to and physical dependence on morphine in the rat. The tolerance to the analgesic and hyperthermic effects of morphine was demonstrated by decreased responses in the rats implanted with morphine pellet in comparison to the placebo-treated controls. The physical dependence was shown by the greater weight loss after removal of the pellet in the rats implanted with morphine pellets when compared to rats implanted with placebo pellets. The pellets were removed (withdrawn) and, after 6–8 h, the rats were sacrificed and the cerebral cortex was isolated. In another experiment the pellets were left in place (tolerant-dependent rats). The 5-HT₁ and 5-HT₂ receptors were characterized by using [^3H]5-HT and [^3H]spiroperidol as the ligands and unlabelled 5-HT and ketanserin, respectively, to determine non-specific binding. The [^3H]5-HT bound to 5-HT₁ receptors on membranes from the cerebral cortex of rats implanted with placebo pellets, at a single high affinity site, with a B_max of 102 ± 10 fmol/mg protein and a K_d of 6.02 ± 0.98 nM. Implantation of morphine pellets, followed by removal of the pellets resulted in a 50% increase in the B_maxvalue of [^3H]5-HT but the K_d values did not change. In rats from which the pellets were not removed, the B_max and K_d values of [^3H]5-HT in placebo- and morphine-treated groups did not differ. [^3H]Spiroperidol bound to 5-HT₂ receptors on cortical membranes of rats implanted with placebo pellet at a single high affinity site with B_max and K_d values of 131 ± 5 fmol/mg protein and 0.22 ± 0.01 nM, respectively. The implantation of pellets of morphine followed by removal of the pellets did not alter the characteristics of 5-HT₂, receptors, however in rats with the pellets in place, the B_max for 5-HT₂ receptors in placebo- and morphine-treated groups did not differ but the K_d values were much smaller in morphine-treated rats compared to rats implanted with placebo pellets. It is concluded that the development of tolerance to, and physical dependence on, morphine by implantation of pellets results in up-regulation of 5-HT₂ receptors whereas in morphine-abstinent rats there is a selective up-regulation of 5-HT₁ receptors on the membranes in the cerebral cortex.     

Light-dark differences in [3H]-paroxetine binding to rabbit platelet membranes

Summary [³H]-Paroxetine binding to rabbit blood platelet membranes from samples obtained under light and dark conditions was examined. Animals were kept on a 14 h light (L) — 10 h dark (D) schedule and blood samples were collected at L + 7 and D + 5 h. Significant differences were found for B _max values of [³H]-paroxetine binding, with low B _max values during the light period and high B _max values during the dark period. The K _d values were not significantly different. These results confirm previous observations on light-dark differences of [³H]-imipramine binding in rabbit blood platelets suggesting the existence of a circadian rhythm for the 5-HT transporter complex.Send offprint requests to S. Z. Langer at the above address     

3H-Imipramine binding and serotonin uptake in platelets from untreated depressed patients and control volunteers

Human platelets possess specific high-affinity binding sites for ³H-imipramine which have similar characteristics to the sites previously described in human and animal brain. In a group of untreated depressed patients, the B_max of ³H-imipramine binding and the V _max of serotonin uptake in their platelets were found to be significantly lower than in a group of control volunteers. There was no significant difference in the K _d values for ³H-imipramine binding but the K _m values of ³H-serotonin uptake were decreased in the depressed patients. When the measurements of ³H-imipramine binding and ³H-serotonin uptake were compared in the same individual, however, there was no correlation between the individual B _max and V _max values or the K _d and K _m values. These results suggest that although the ³H-imipramine binding site and the mechanism for serotonin uptake are associated, they are not identical.     

Disposition of [3H]fluvastatin following single oral doses in beagle dogs and rhesus monkeys with bile fistulae

The disposition of [³H]fluvastatin was examined following single oral doses in dogs (12.4 mg kg^?1) and monkeys (0.48 45.5 mg kg^?1) with bile fistulae. Serial plasma and complete urine, feces, and bile were collected at designated intervals for 3 or 4 d, and were analyzed for total radioactivity and unchanged fluvastatin. In the dog, peak radioactivity concentrations (C_max) averaged 7260 ng equiv. mL^?1 and the mean time to peak (t_max) was ～ 9 h. In the monkey, the mean radioactivity t_mzx values were ～ 5 and 13 h following the low and high doses, the respective C_max values being 116 and 10400 ng equiv. mL^?1. The mean AUC of total radioactivity was proportional to the dose while that of fluvastatin was overproportional to dose, suggesting dose indepedent absorption but saturable first-pass effect. The AUC ratio of unchanged fluvastatin versus total radioactivity was approximately 63% in the dog, and 9% and 13% for the low and high doses, respectively in the monkey. The bile was the major excretory route of radioactivity (dog, 56%; low-dose monkey, 73%; high-dose monkey, 69%) whereas the renal pathway accounted for < 5% of the dose in both species. Approximately 12% of the biliary radioactivity in the dog was due to intact fluvastatin, compared with 0% and 7.5% after the low and high doses in the monkey. These results showed a smaller extent of fluvastatin metabolism in the dog than in the monkey, and suggested that metabolism in the monkey was saturable in the dose range studied.     

3H-Flunitrazepam binding to bovine retina and the effect of GABA thereon

Bovine retinae were examined for their ability to bind ³H-flunitrazepam. Whole retinal homogenates revealed specific, high affinity, and saturable ³H-flunitrazepam binding (K_d, 1.1 nM; B_max, 19.5 fmoles/mg tissue). In washed membrane preparations, 10 μM GABA caused a dramatic decrease in the K_d (42 percent). The rank order of potency for the inhibition of ³H-flunitrazepam binding by different benzodiazepines was: clonazepam > clobazam ? Ro 5-4864. These results are supportive for the presence of benzodiazepine receptors in the bovine retina and indicate a likeness with the previously described benzodiazepine receptors of mammalian brain.     

Changes in relative potency among positive GABA<Subscript>A</Subscript> receptor modulators upon discontinuation of chronic benzodiazepine treatment in rhesus monkeys

Rationale Benzodiazepine treatment can result in dependence as evidenced by signs of withdrawal upon discontinuation of use. Objective Positive GABA_A receptor modulators were examined for their capacity to attenuate flumazenil-like discriminative stimulus effects (i.e., withdrawal) that emerge upon discontinuation of chronic benzodiazepine treatment. Methods Rhesus monkeys receiving chronic diazepam (5.6 mg⁻¹ kg⁻¹ 24 h⁻¹ p.o.) discriminated flumazenil (0.1 mg/kg s.c.) from vehicle. Results Upon discontinuation of diazepam treatment, responding switched from the vehicle to the flumazenil lever, although at different times among monkeys. The shorter-acting benzodiazepine lorazepam (3.2 mg⁻¹ kg⁻¹ 8 h⁻¹) was substituted for diazepam and, 11 h after lorazepam, monkeys consistently responded on the flumazenil lever. Flumazenil-lever responding after acute lorazepam deprivation was attenuated not only by benzodiazepines (lorazepam and midazolam) but also by positive GABA_A receptor modulators acting at neuroactive steroid (pregnanolone and alfaxalone) and barbiturate sites (pentobarbital). Deprivation-induced responding on the flumazenil lever was not attenuated by low efficacy positive GABA_A modulators (bretazenil and L-838,417) or non-GABA_A receptor ligands (ketamine and cocaine). Neuroactive steroids were relatively more potent than other positive GABA_A receptor modulators in attenuating deprivation-induced flumazenil-lever responding, as compared to their relative potency in monkeys discriminating midazolam and otherwise not receiving benzodiazepine treatment. Conclusions These results suggest that positive GABA_A receptor modulators acting at different sites attenuate withdrawal induced by discontinuation of benzodiazepine treatment, consistent with previous studies suggesting that the same compounds attenuate flumazenil-precipitated withdrawal. Differences in the relative potency of positive modulators as a function of acute versus chronic benzodiazepine treatment suggest that neuroactive steroids, in particular, are especially potent in benzodiazepine-dependent animals.