fIivIoPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.4 305

Characterization of the Binding and Comparison of the Distribution of Benzodiazepine Receptors Labeled with pH1Diazepam and [3H]Alprazolam l&s K. Wamsley, Ph.D., Lisa L. Longlet, B.S., MaryAnne E. Hunt, Ph.D., Donald R. Mahan, IfiMilrio E. Alburges, Ph.D.

Ttrbinding characteristics of [3H]diazepam and benzodiazepine drugs and apparently do not overlap onto �prazolam were obtained by in vitro analysis of other subtypes of receptors. These experiments were IIfiorrsof rat brain. Dissociation, association, and performed by both binding assay in tissue sections and by sDlItionaTUllyses were performed to optimize the light microscopic autoradiography. The major difference GdilWnsfor obtaining selective labeling of between the labeling of the two compounds is· represented llaldiDzepinerecept ors with the two tritiated by the peripheral benzodiazepine sites, which are GIIpOUnds. Both drugs approached equilibrium rapidly recognized by [3H]diazepam, but not occupied by nntro. Rosenthal analysis (Scatchard plot) of the [3H]alprazolam (at nanomolar concentrations). This amiondata indicated a similar finite number of difference was readily apparent in the auto radiograms. IIII;otSwas being occupied by both ligands. Other pharmacokinetic or pharmacodynamic properties Clapetitionstudies, using various ligands to inhibit both must distinguish these two benzodiazepines. fH}bu.tpam and [3H]alprazolam indicated that these [Neuropsychopharmacology 8:305-314, 1993J tIIIcmnpoundsbind to the tissue sections as typical

lIT WORDS: Benzodiazepine receptors; Anxiolytics; in the treatment of panic disorder (Feighner et a1. 1983; 0upmI;Alprazolam; Autoradiography; Localization; Pitts et a1. 1983; Shehi and Patterson 1984; Rickels et Drtsity;Binding characteristics a1. 1985; Alexander and Alexander 1986; Leibowitz et 1986; 1986), A¥azolamis a triazolobenzodiazepine that is distinct al, Mendels and SchIess whereas many DIIother cornpounds in its class due to its unique clin­ other typical benzodiazepines are not as efficaciousin g profIle (Fawcett and Kravitz 1982; Dawson et a1. these latter two areas, but are remarkably effective 114;Cir aulo et a1. 1986; Dunner et a1. 1986). Alprazo­ anxiolytics (others are used as sedative-hypnotics or an­ .. hasbeen used as an anxiolytic, antidepressant, and ticonvulsants). Results from several studies have sug­ gested alprazolam may be recognizing other receptor sites in addition to benzodiazepine receptors (Cer­ fan the Neuropsychiatric Research Institute, Fargo; and the nansky et al, 1982; Sethy and Hodges 1982; Charney tsof Pharmacology and Neuroscience, University of North IIIfIrtmen and Heninger 1985; Eriksson et al, 1986; Kostowski et 1Iika.School of Medicine, Grand Forks and Fargo, North Dakota; alDIeUp john Company, Kalamazoo, Michigan. al, 1986; Soderpalm and Engel 1989), in the central ner­ Addressre print requests to: James K. Wamsley, Ph.D., Director vous system (CNS). The in vitro analysis of [3H]al­ d_ScienceResearch, Neuropsychiatric Research Institute, 700 prazolam binding in synaptosomal preparations of rat "Avenue South, Fargo, North Dakota 58103. IDivedSept ember 3, 1991; revised March 17, 1992; accepted April brain has not been able to verify this as a direct effect -\J9I12. (McCabe et a1. 1990). To analyze the potential for over-

• n93American College of Neuropsychopharmacology Mished by Elsevier Science Publishing Co., Inc. 5Avenue of the Americas, New York, NY 10010 0893-133X/93/$6.00 NEUROPSYCHOPHARMACOLOGY VOL. 306 J.K. Wamsley et ai. 1993- 8, NO.�

lap of [3Hlalprazolam onto other sites, a receptor au­ incubation of groups of sections in various concenm toradiographic approach (Kuhar et al. 1985) was used tions of the radioactive ligands to establish saturatioa in the present study. Previous investigations, aimed at Competition studies were performed by labeling sec· analyzing the distribution of benzodiazepine receptors tions with a 2-nmollL concentration of the radiolabel� in the CNS, have principally involved the use of ligand using the parameters established in the previ­ pHlflunitrazepam as the ligand of choice (Young and ously outlined experiments, in the additional presena Kuhar 1979, 1980). Relatively little information is avail­ of 10-4 to 10-11 mollL concentrations of the follo� able on the binding of [3Hldiazepam after its initial compounds: clonidine, CL218,872, sulpiride, desipra­ characterization in membrane preparations (Braestrup mine, imipramine, 4-0H alprazolam, or flurazepant and Squires 1977; Gallager et al. 1981). We sought to The ICso values were obtained by plotting the percent use a typical anxiolytic compound for comparison with of radioligand bound versus the percent of radioligand alprazolam and to provide the binding conditions and bound multiplied by the molar concentration of the characteristics for obtaining selective labeling of both competing ligand. In all cases, adjacent sections wert [3Hldiazepam and [3H]alprazolam to slide-mounted incubated in the presence of 10-6 mollL clonazepam tissue sections for autoradiographic analysis. to establish nonspecifIc binding. For the autoradiographic studies, slide-mounted sections (20 J.1 in thickness) of whole brain were labeled MATERIALS AND METHODS using the optimum binding conditions and, insteadd wiping the sections from the slide, the sections wert Male Long-Evan's rats (weighing between 180 and 200 dried by blowing cool dry air over the tissue surface, gm) were deeply anesthetized with C02 and perfused These sections were subsequently exposed to tritium intracardially with ice-cold isotonic saline. The brain tis­ sensitive film (Amersham Hyperfilm, Arlington sues of the animals were rapidly dissected free from Heights, IL). After an appropriate exposure period, the surrounding structures and frozen onto microtome fIlms were developed and analyzed by computerized chucks by slow immersion in isopentane at -70°C. Sec­ microdensitometry (Palacios et al. 1981) using anMOD tions, 10 J.1 in thickness, either frontal or sagittal plane, system (Imaging Systems Corporation, St. Catherines, were cut on a cryostat microtome and thaw-mounted Ontario). Tritium standards (Amersham, Arlington onto subbed microscope slides. The brains of some Heights, IL) were included in the exposure of each DIm animals were removed from the skull and gently to quantitate femtomoles of ligand bound per milligram homogenized in a glass tube with a Teflon-coated pes­ tissue. Another group of sections was incubated ina tle. Frozen sections of this homogenate were cut in the Kd concentration of the radioactive ligand (inan effort cryostat as before. This procedure assured a uniform to occupy a major portion of the sites recognized bylbe distribution of different cell types in each section so radioactive compound) in the additional presence ofjn. more consistent data could be obtained. Alltissues were creasing concentrations of the other benzodiazepine, stored overnight in the presence of desiccant prior to Thus, incubations were performed with [3Hldiazepam being utilized in the incubation procedures. in the presence of increasing concentrations of unla­ For the biochemical assays, slide-mounted tissue beled alprazolam and [3Hlalprazolam was used to la­ sections of homogenized brain were initially given a bel sections inthe presence of nonradioactive diazepam. 3-minute preincubation period in distilled water to os­ These sections were analyzed by receptor autoradiog· motically disrupt the cells and release any gamma raphy to determine if the radioactive compound oc· aminobutyric acid (GABA) or other constituents that cupied sites where the binding was not inhibited by the may interfere with the subsequent binding of the ra­ other benzodiazepine. dioactive ligand (McCabe et al. 1988). This was followed by two 5-minute rinses, after which individual groups of sections were incubated for 60 minutes in the pres­ RESULTS ence of the radioactive ligand in Tris HCl buffer (0.17 mollL, pH 7.6) at between 0 and 4°C. Initially, a 2 Comparison of the binding characteristics of [3H]al· nmollL concentration of pHlalprazolam (15 to 45 prazolam and [3Hldiazepam to slide-mounted tissue Ci/mmol, supplied by the Upjohn Company, Kalama­ sections of rat brain indicated many similarities. The zoo, MI) or [3Hldiazepam (85.2 Ci/mmol, Dupont initial osmotic shock of the slide-mounted tissue sec· NEN, Boston, MA) was used to label the sections. The tions dramatically reduced the binding of pH]alprazo. rinse time was varied and the tissue sections were wiped lam. The magnitude of this reduction in binding was from the microscope slides with fIlter paper and the as high as 50% and affected only specifIc binding bound radioactiVity determined by liquid scintillation (nonspecifIcbinding remained unchanged). Bindingd counting. Next, the incubation time was varied, with [3Hldiazepam was also reduced by the osmotic shock, the rinse time held constant. This was followed by the but not to the extent of the alprazolam binding. The Nn!aoPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.4 Comparison of [3H]Diazepam and [3H]Alprazolam 307

II!beIingwithboth ligands was temperature dependent Fig. 1). A BMAX of 189.8 and 195.7 fmol of receptor mdaJI subsequent experiments were performed at ice­ bound per milligram tissue was obtained for [3H]al­ WIte mperature. Analysis of the dissociation data (Fig. prazolam and [3H]diazepam, respectively. Inhibition ,wasperformed using a nonlinear regression program curves (Fig. 2) generated by incubating various com­ �h Pad), whichresulted in a calculated K-1 (Bylund pounds in the presence of the two radioactive ligands, /lidYamamura 1990) of 0.034 minute-1 for alprazo­ again indicated similar sites were being recognized by II1II.From the association data it could be established both of these compounds. The inhibition curves for .. equilibrium was achieved with [3H]alprazolam flurazepam, 4-hydroxyalprazolam, CL218,872, and di­ \11.1)after a 20-minute incubation period. The K1 cal­ azepam against [3H]alprazolam are shown in Figure 2. CIlIated for alprazolam was 0.0075 min -l/nmollL -1 The inhibition constant (Ki) for each substance is indi­ pmvidinga kineticallyderived estimate of the Kd equal cated in Table 1. m 4.5 nmollL. Similar analyses were performed for Autoradiographic analysis of the binding sites oc­ �epam (not shown). Analysis of saturation cupied by diazepam and alprazolam showed many iIIIherms(using a rectangular hyperbola) best fIta one­ regions of overlap (Fig. 3 and Table 2). The highest den­ _modelwith a Kct of 3.2 nmollL for [3H]alprazolam sities were found in the cerebral cortex, hippocampus, /lida 6.2 nmollL for [3H]diazepam. These fIgures are and molecular layer of the cerebellum and substantia dFtlyl ower than those obtained from Scatchard anal­ nigra. Somewhat lower densities were found in many JIis(Rosenthal plot) of the saturation curve data (see thalamic and hypothalamic regions as well as at the cau-

A B

1OCO 700 • total • total 600 • epecIfIc • epecific 500 .. nonapeclflc 2 � • a.. � 0 300

200

2CO 100

0 30 � 60 90 0 2 " 6 8 10 15 75 TIME (MINS) TIME (MINS)

C 0

t2 50 Kd = 4.00 • total to ..0 = W • 189.8 .. nonepecific W B.... e 0:: • 30 • epecific ...... LL e 0 Z 20 II ::J 0 • CD 10

0 0 25 60 75 100 125 150 175 200

[�]ALPRAZOLAM [nM] BOUND

1p21. Graphs illustrating characterization of [3H]alprazolarnbinding. A = dissociation; B = association; C = satura­

c.;lndO= Rosenthal (Scatchard) plot of [3H]alprazolam binding to slide-mounted tissue sections of rat brain. The Rosen- 6llplot-derived K.:t is 4.0 nmol with a Bmax of 189. 8 fmol of receptor bound/mg tissue. Similar analysis of [3H]diazepam Nw!da I

centrations used in this study. This was quite remark· • 4-0H Alpraz able on the autoradiograms generated by inhibiting the A CL 218872 binding of one compound with the other (Fig. 4). In • Diazepam those sections labeled with [3H]diazepam in the pres­ • Flurazepam ence of alprazolam, high concentrations of the latter compound inhibited allof the diazepam binding exce� 50 that in regions of the peripheral sites. Under the oppo­ site condition, all of the sites occupied by alprazolam were inhibited by diazepam. No other regions of labeJ. ing could be shown to be statistically different.

o������������� -11 -10 -9 -8 -7 -6 -5 -4 -3 DISCUSSION log [M] The benzodiazepines represent a diverse set of com­ Figure 2. Graph illustrating selectivity of [3H]alprazolam pounds all with many similar characteristics (Haefely binding. The ability of several ligands to compete for the sites 1989; Williams and Olsen 1989). In fact, it has been recognized by [3H]alprazolam was analyzed in tissue sec­ tions. Ligands with reasonable affmity for the sites occupied pointed out that since many of these compounds share by [3H]alprazolam included 4-hydroxyalprazolam (an al­ the same long-acting metabolites, they may indeed be prazolam metabolite), diazepam, CL218,872, and flurazepam. similar in their pharmacodynamics (Arendt et al. 19Sn. The BZl receptor selective agent CL218,872 (a triazolopyrida­ The triazolobenzodiazepine, alprazolam, appears to be zine) inhibited the binding in a very shallow manner indicat­ unique on the basis of its clinical pro&le since it, as o� ing the recognition of more than one site of the parent com­ posed to other benzodiazepines, appears to be useful pound. in the treatment of depression and panic disorder (Dawson et al. 1984). The binding of [3H]alprazolam however, looks very similar to [3H]diazepam (McCabe date putamen and nucleus accumbens. A similar dis­ et al. 1990). Both label a fmite receptor population that tribution was found with both ligands except that the shows the characteristics of benzodiazepine receptors. compounds differed in their ability to recognize the pe­ The kinetics of the binding with the two ligands are simi­ 3 4). ripheral benzodiazepine sites (Figs. and Diazepam lar and subject to modulation by GABA since osmon: quite readily labeled these sites in the ependymal lin­ shock removed some of the specifIc binding in each ing of the ventricles, the choroid plexus, and other case. Competition of one compound against the other regions where the blood-brain barrier is permeable. AI­ also supports the conclusion that the binding is taking prazolam, however, did not label these sites at the con- place at the same sites. Several studies have indicated an apparent over· lap of alprazolam's effectsonto other neurotransmitter Table 1. Competition of [3H]Alprazolam Binding systems including J3-adrenergic, a-adrenergic, and Ligand Ki [nM)a dopaminergic receptors. The analysis of [3H]alprazo. lam binding in synaptosomal preparations provided 00 >60,000 Clonidineb indication of a direct interaction between the benzodi­ CL218,872 31.29 azepine and these adrenergic receptor subtypes (Mc· Sulpirideb >60,000 Desipramineb >60,000 Cabe et al. 1990). Testing of many other compounds Imipramineb >60,000 against the binding of [3H]alprazolam also indicated Diazepam 7.46 there was not any overlap onto sites outside of the ben­ 38.62 4-0H Alprazolam zodiazepine receptors. Flurazepam 6.12 Pharmacologic studies have indicated the presence a The Ki values were computed by calculation of the ICso from the of at least three subtypes of benzodiazepine receptors inhibition curves, using Graph Pad software (Bylund and Yamamura, in the CNS (Klepner et al. 1979; Nielsen and Braestrup 1990). The experiments were performed in triplicate and each sam­ ple was repeated three times; the results are expressed as the mean 1980; Sieghart and Karobath 1980; Braestrup an dNieJ. of these experiments. Tissue sections were selected from four differ­ sen 1981; Hirsch et al. 1982; Squires 1983). Theseare ent animals and the data pooled. The sections were wiped from the the BZ1, BZ2, and peripheral benzodiazepine sites that slides and the residual radioactivity remaining bound to the tissue are also known as <01- , respectively (Langer andAr· was determined by ligand scintillation counting. 3 b The antidepressant imipramine (selective for 5-HT uptake), billa 1988). The so-called peripheral benzodiazepine clonidine (an a2-adrenergic receptor agonist), sulpiride (a DA2 sites have been discovered in the brain (Benavides eI receptor antagonist), and desipramine (an antidepressant selective for norepinephrine uptake) did not compete for [3Hlalprazolam al. 1983b; Gehlert et al. 1983, 1985; Schoemaker eta!. binding. 1983; Anholt et al. 1984) and these sites appear to have tin.'IOPSYCHOPHARMACOLOGY VOL. NO. 4 1993- 8, Comparison of [3H]Diazepam and [3H]Alprazolam 309

A

Figure 3. Autoradiograms of diazepam and alprazolam binding. Photomicro­ graphs of the actual autoradiograms of sagittal sections of rat brain labeled with [3H]diazepam (A) and [3H]alprazolam (B) binding. Note the similarity in the regions Tu AA showing the presence of autoradiographic grains (black against the white back­ ground). Although alprazolam binding is much lighter, the distribution is the same B as that for diazepam except in the area of the lateral ventricle (choroid plexus). The exposure period for [3H]alprazolam was already 4 months. Doubling this time would be expected to generate an autora­ diogram similar to A. Thiscan be performed by computer enhancement to compare the relative densities as seen in Figure 4. Ab­

breviations: OB = olfactory bulb; IV = lam­

ina IV of the cerebral cortex; LV = lateral

ventricle; H = hippocampus; DG = den­ tate gyrus; SuG = superficialgrey layer of

the superior colliculus; IC = inferior collic­

ulus; Tu = olfactory tubercle; AA = amyg­ AA daloid area; SN = substantia nigra.

aaniquesubcellular distribution (Anholt et al. 1986; Ba- affinity GABAA sites (Unnerstall et al. 1981; Wamsley 6andSkolnick 1986). Some of the centrally acting ben­ and Palacios 1984; McCabe and Wamsley 1986). �pines recognize these sites whereas others do The results of the present study support the con­ �. Nanomolar concentrations of agents such as di- clusion that [3H]alprazolam recognizes those sites la­ 5Ipine andflunitrazepam will bind detectably to the beled with [3H]diazepam. Computerized microdensito­ peripheralsi tes whereas clonazepam, alprazolam, tri­ metric analysis of the autoradiographic fIlmsgenerated !dam, fluraze by the two ligands show only relative differencesin the p.iem, do not. Most benzodiazepines recognize the amount of labeling in individual regions of the brain *"two subtypes of benzodiazepine receptor (BZ1 where benzodiazepine receptors exist. By standardiz­ !lidBZ2) with equal high affInity. However, the ben­ ing these measurements to a central area, no statisti­ !lldiazepine, quazepam, and the �-carbolines as well cally differentamount of labeling could be obtained by NteveraInonbenzo diazepine compounds (CL218,872, one ligand versus the other, even though the labeling Sllpidem, and CGC91164) preferentially bind the BZ1 of diazepam is relatively higher in each individual area. mptor(Klepner et al. 1979; Lippa et al. 1979; Nielsen Thus, it would appear that the receptor sites recognized !lidBraestrup 1980; Iorio et al. 1984; Wamsley et al. by [3H]alprazolam and [3H]diazepam are the typical 115; Billard et al. 1987). This receptor subtype also benzodiazepine receptors described in classic studies. lIKIwsaunique distribution in various nuclei of the CNS Using conditions that provide optimum signal-to­ (Younget al. 1981; Wamsley et al. 1985; Niddam et al. noise binding ratios, it was possible to create competi­ tI'J;Yezuita et al. 1988). Both receptor subtypes ap­ tion between one of the radioactive ligands against the ptmto be allosterically bound to GABA receptors and other. As higher and higher concentrations were \heyshow a distribution similar to that of the high- reached, it should have been possible to demonstrate 310 J.K. Wamsley et aI. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.

Table 2. Regional Distribution of [3H]Diazepam and [3H]Alprazolam Binding Site in Rat Brain"

Bound (fmoIlmg Tissue ± SEM)

Brain Area [3H] Diazepam [3H]Alprazolam

Frontoparietal Cortex Laminae I-III 193. 70 ± 7. 51 165.17 ± 8.25 Laminae IV 217. 51 ± 5. 44 206. 38 ± 5.91 Laminae V-VI 159. 64 ± 4.33 156. 90 ± 5. 18 Caudate Putamen 84.66 ± 2.43 76. 92 ± 3.20 Globus Pallidus 57.56 ± 2. 77 50.25 ± 5.28 Ventral Pallidum 95.87 ± 5.77 87.82 ± 8.32 Field CAl of Ammon's Horn 155.77 ± 2.46 167. 21 ± 4.47 Dentate Gyrus 182.67 ± 10.26 189. 10 ± 6. 30 Ventral Posterolateral/ Ventral Posteromedial Thalamic Nuclei 71.97 ± 7. 96 58.25 ± 4.16 Zona Incerta, dorsal/ventral 90.41 ± 7. 96 94.92 ± 3. 82 Substantia Nigra 113.66 ± 6.26 114.77 ± 6.08 Superior Gray Layer of the Superior Colliculus 218.53 ± 16.33 182.22 ± 5.61 External Cortex of the Inferior CoIIiculus 152.36 ± 22.87 100. 68 ± 7. 26 Medial Geniculate Nuclei dorsal/ventral 90.72 ± 6.11 91.29 ± 10.17 Entorhinal Cortex 201.99 ± 14.22 161.13 ± 37.50 Microcellular Tegmental Nucleus 171. 21 ± 23. 86 150. 56 ± 16.12 Molecular Layer of the Cerebellum 120. 70 ± 3.02 122.21 ± 7.95 Granular Layer of the Cerebellum 29.30 ± 4.32 44.88 ± 8.86 Accumbens Nucleus 137. 65 ± 9.50 121.70 ± 6.05

" The data are expressed as the mean ± SEM in femtomoles of ligand bound per milligram of tis­ sue. These values were obtained from digitized images of the autoradiograms and standards on the MCDI imaging system. Only relative differences can be seen. These values are not statistically sig­ nificant from each other. p < 0.01.

labeling of the radioactive compound outside of the (Fawcett and Kravitz 1982; Sethy and Harris 1982a;St realm of sites recognized by the un1abeled compound, thy 1983). For instance, low doses of alprazolam aPPII ifsuch nonselective bindingwere occurring. Indeed this ently result in an increase of benzodiazepine receplll was demonstrated by the inability of alprazolam to in­ numbers, rather than a decrease (Miller et al. 1987)1. hibit [3H]diazepam binding from the peripheral bind­ seen with higher doses (Sethy and Harris 1982b).1t ing sites in the choroid plexus and the ependymal lin­ terestingly, the peripheral benzodiazepine bindingsilll ing of the ventricles. This appeared to be the on1y have been associated with various effects includq difference between the two ligands that we could ascer­ production of steroid hormones, renal hypertension tain in our microscopic analysis. The relative importance and an interaction with anesthetic binding sites (W� of the latter observation in reference to the unique clin­ et al. 1984a,b; Eriksson et al. 1986; Mestre et al.l98h ical profIle of alprazolam is unknown. This could be Clark and Post 1990; Massotti et al. 1990; Papadopoub meaningful, however, since there are many hypothe­ et al. 1990). How these effects relate to the centralmem ses regarding the potential roles the peripheral sites may anisms involved in the potential actions of compound! play in biologic function coupled with the widespread acting at these sites remains to be' determined. Likf. appearance of these receptors in many tissues (Bena­ wise, potential species differences in benzodiazepin! vides et al. 1983a; Le Fur et al. 1983; Quirion 1984; Wang receptors may preclude our ability to extrapolate Oil! et al. 1984a,b; De Souza et al. 1985; Mestre et al. 1986; fmdings across phylogeny. Starosta-Rubinstein et al. 1987; Verma et al. 1987). It appears likely that alprazolam's effects on other There are several other possible explanations for systems occur "downstream" rather than as a dira1 alprazolam's effects that involve pharmacodynamic receptor-mediated phenomenon. There is a potential differences with other benzodiazepine compounds however, for diazepam to affect the peripheral sit!! NEUIOPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.4 Comparison of [3H]Diazepam and [3H]Alprazolam 311

1f!Ie4. Comparison of [3H]alprazolam and [3H]diazepam receptor autoradiography. (A) Computer-generated images illllloradiogramsproduced by labeling sagittal sections of rat brain with (A) [3H]diazepam or (B) [3H]alprazolam. The color Dtwas held constant to show the level of binding in each case. The specific activity of the two ligands, however, makes .dftctcompar ison difbcult. In (B) and (C), the color scale was adjusted to artificially simulate the colors across the two Idilns.The relative differencesbetween colors on the same section was not changed, so the binding can now be compared. NaIrthe similarity between the sites recognized by each ligand. The section shown in (E) was labeled with [3H]diazepam Glhtpresenceof 10-8 mol alprazolam. In (F), the section was labeled with [3H]alprazolam in the presence of diazepam (ltlmol). Note the similarities in the ability of each ligand to compete for the areas occupied by the other compound. c.exceptionis readily noted in the fourth ventricle (LR4V). This area contains the peripheral sites recognized by diaze­ filii.but n ot by low concentrations of alprazolam. Thus, these sites are bound by the radioactive drug, but not inhibited olE).They are un recognized by the radioactive compound in (F). Abbreviations: OB = olfactory bulb; IV = lamina IV dlhtcerebralcortex; LV = lateral ventricle; H = hippocampus; DG = dentate gyrus; SuG = superfIcial grey layer of the �r colliculus; IC = inferior colliculus; Tu = olfactory tubercle; AA = amygdaloid area; SN = substantia nigra. 312 J.K. Wamsley et al. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.1

directly, as well as BZl and BZ2 receptors, which could 2-oxo-quazepam for BZ1 binding site and demonstratD distinguish it from alprazolam, but not presumably of H-2-oxo-quazepam as a BZ1 selective radioligand.li Sci 42:179-187 other benzodiazepines like clonazepam. Molecular bio­ logic studies indicate the presence of more than just Braestrup C, Squires RF(1977): SpecifIcbenzodiazepi nerec£t tors in rat brain characterized by high affinity [3H]dim three benzodiazepine receptor subtypes due to the pam binding. Proc Nat! Acad Sci USA 74:3805-3809 identifIcationof several distinct subunits of the GABA Braestrup C, Nielsen M (1981): [3H]-Propyl-l3-carboline-� receptor (Liiddensand Wisden 1991). Drugs previously carboxylate as a selective radioligand for the benzodialt BZl classifIed as selective have been shown to interact pine receptor subclass. J Neurochem 37:333-341 differently with the a-GABAA receptor subunit and Bylund DB, Yamamura HI (1990): Methods for receptorbiJld. other variants, providing a new level of differentiation ing. In Yamamura HI, Enna SJ, Kuhar MJ (eds), Methlll of benzodiazepine compounds. Alprazolam and diaze­ in Neurotransmitter Receptor Analysis. New York, Hi pam may have unique profIles in relation to these ven Press, pp 1-35 subunits that could account for their unique properties. Charney OS, Heninger GR (1985): Noradrenergic functilrl Future experimentation in this area will be required to and the mechanism of action of antianxiety treatment resolve these issues. Arch Gen Psychiatry 42: 458-467 Ciraulo CA, Barnhill JG, Boxenbaum HG, Greenblatt III Smith RB (1986): Pharmacokinetics and clinical effectso' alprazolam following single and multiple oral doses! ACKNOWLEDGMENTS patients with panic disorder. J Clin PharmacoI4: 292-N Clark M, Post RM (1990): Lidocaine binds with high affmitJ The authors would like to thank Karen Johnson for her to peripheral-type benzodiazepine receptors. Eur JPhs­ secretarial assistance, Cindy Belohlavek and Candy Johnson macol 179:473-475 for technical assistance, and Diane Nordeng for her help in the library. This research was supported by funds from the Csernansky JG, Csernansky CA, Hollister LE (1985): [311f Upjohn Company. Sulpiride labels mesolirnbicnon-dopaminergic sitesthi bind antidepressant drugs. Experientia 41:1419-1421 Dawson GW, Jue SG, Brogden RN (1984): Alprazolam: Alfo view of its pharmacodynamic properties and e£fi.cacyiD REFERENCES the treatment of anxiety and depression. Drugs 11: 132-147 Alexander PE, Alexander DO (1986): Alprazolam treatment De Souza EB, Anholt RRH, Murphy KMM, Snyder SH, Ku­ for panic disorders. J Clin Psychiatry 47:301-304 har MJ (1985): Peripheral-type benzodiazepine receptOl! Anholt RRH, Murphy KMM, Mack GE, Snyder SH (1984): in endocrine organs: Autoradiographic localizationinri Peripheral-type benzodiazepine receptors in the central pituitary, adrenal and testis. Endocrinology 116: 567-51l nervous system: Localization to olfactory nerves. J Neu­ Dunner DL, Ishiki 0, Avery DH, Wilson LG, Hyde TS (1986): rosci 4:593-603 Effectof alprazolam and diazepam on anxiety and pani Anholt RRH, Pedersen PL, De Souza EB, Snyder SH (1986): attacks in panic disorder: A controlled study. J OinPsy· The peripheral-type benzodiazepine receptor: Localiza­ chiatry 47:458-460 tion to the mitochondrial outer membrane. J BioI Chern Eriksson E, Carlsson M, Nilsson C, SoderpalrnB (1986): Does 261: 576-583 alprazolam, in contrast to diazepam, activate alpha 2-adrenoceptors involved in the regulation of rat growlh Arendt RM , Greenblatt OJ, Liebisch DC, Luu MD, Paul SM (1987): Determinants of benzodiazepine brain uptake: hormone secretion? Life Sci 16:1491-1498 lipophilicity versus binding affinity. Psychopharmacol­ Fawcett JA, Kravitz HM (1982): Alprazolam: Pharmacokinet­ ogy 93:72-76 ics, clinical efficacy, and mechanism of action. Phar· Basile AS, Skolnick P (1986): Subcellular localization of macotherapy 2:243-254 «peripheral-type» binding sites for benzodiazepines Feighner JP, Aden GC, Fabre LF, Rickels K, Smith WT(1983� in rat brain. J Neurochem 46:305-308 Multiclinic double-blind safety and efficacycomp arisoll Benavides J, Malgouris C, Imbault F, Begassat F, Uzan A, of alprazolam, imipramine, and placebo in the treatmem Renault C, Dubroeucq MC, Gueremy C, Le Fur G (1983a): of depression. JAMA 249:3057-3064 "Peripheral-type" benzodiazepine binding sites in rat Gallager OW, Mallorga P, Oertel W, Henneberry R, Tallman adrenals: Binding studies with [3H]PK11195 and au­ J (1981): [3H]Diazepam binding in mammalian centri toradiographic localization. Arch Int Pharmacodyn Ther nervous system: A pharmacological characterization.) 266:38-49 Neurosci 1: 218-225 Benavides J, Quarteronet 0, Imbault F, Malgouris C, Uzan Gehlert DR, Yamamura HI, Wamsley JK (1983): Autorad» A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G graphic localization of "peripheral" benzodiazepinebind­ (1983b): Labelling of "peripheral-type" benzodiazepine ing sites in the rat brain and kidney using [3H]R05-4864. binding sites in the rat brain by using [3H]PK-11195, an Eur J Pharmacol 95:329-330 isoquinoline carboxamide derivative: Kinetic studies and Gehlert DR, Yamamura HI, Wamsley JK (1985): Autorad» autoradiographic localization. J Neurochem 41: 1744-1750 graphic localization of peripheral-type benzodiazepine Billard W, Crosby G, Iorio L, Chipkin R, Barnett A (1987): binding sites in the rat brain, heart and kidney. Naunyn Selective affinityof the benzodiazepines quazepam and Schmiedebargs Arch Pharmacol 328:454-460 tinlOPSYCHOPHARMACOLOGY 1993- VOL. 8, NO.4 Comparison of [3HJDiazepam and [3HJAlprazolam 313

a.telyW (1983): The biological basis of benzodiazepine ac­ zodiazepine receptors. Pharmacol Biochem Behav 37: tions.The benzodiazepines today -two decades of re­ 365-370 search and clinical experience. J Psychoactive Drugs Mendels], Schiess AP(1986): Comparative effIcacyof alprazo­ 15:19-39 lam, imipramine, and placebo administration once a day h/ely WE (1989): Pharmacology of the benzodiazepine in treating depressed patients. J Clin Psychiatry 47: receptor. Eur Arch Psychiatr Neurol Sci 238:294-301 357-361 WID, Kochman RL, Sumner PR (1982): Heterogeneity Mestre M, Varriot T, Neliat G, Uzan A, Renault C, Dubroeucq of brain benzodiazepine receptors demonstrated by Me, Gueremy C, Doble A, Le Fur G (1986): PK 11195, [lHl-propyl-p-carboline-3-carboxylate binding. Mol an antagonist of peripheral type benzodiazepine recep­ Pharmacal 21:618-628 tors modulates BAY K8644 sensitive but not f3 or H2- .,LC, Barnett A, Billard W (1984): Selective affInity of receptor sensitive voltage operated calcium channels in I-N-trifluoroethyl benzodiazepines for cerebellar Type the guinea pig heart. Life Sci 39:329-339 I receptor sites. Life Sci 35:105-113 Miller LG, Greenblatt DJ, Barnhill ]G, Deutsch SI, Shader II!pnerCA,Lippa AS, Benson DI, SarnoMC, Beer B (1979): RI, Paul SM (1987): Benzodiazepine receptor binding of Resolution of two biochemically and pharmacologically triazolobenzodiazepines in vivo: increased receptor num­ distinctbenzodiazepine receptors. Pharmacol Biochem ber with low-dose alprazolam. ] Neurochem 49:1595-1601 8ehav11:457-462 Niddam R, Dubois A, Scatton B, Arbilla S, Langer SZ (1987): li!mwskiW,Valzelli L, Baiguena G (1986): Effectof chronic Autoradiographic localization of [3H]-zolpidem binding .dministration of alprazolam and adinazolam on cIoni­ sites in the rat central nervous system. Comparison with dine-or apomorphine-induced aggression in laboratory the distribution of [3HJ-flunitrazepam binding sites. ] rodents. Neuropharmacology 25:757-761 Neurochem 49:890-899 IarMJ(1985): Receptor localization with the microscope. Nielsen M, Braestrup C (1980): Ethyl beta-carboline-3-car­ lnYarnamura HI, Enna SJ, KuharMJ (eds), Neurotrans­ boxylate shows differentialbenzodiazepine receptor in­ mitter Receptor Binding. New York, Raven Press, pp teraction. Nature 286:606-607 !53-176 Olsen RW, McCabe RT, Wamsley]K (1990): GABAA recep­ tor subtypes: Autoradiographic comparison of GABA, ..SZ, Arbilla S (1988): Imidazopyridines as a tool for benzodiazepine, and convulsant binding sites in the rat thecharacter ization of benzodiazepine receptors: A pro­ posaIfor a pharmacological cIassmcation as omega recep­ central nervous system. J Chern Neuroanat 3:59-76 torsubtypes. Pharmacol Biochem Behav 29:763-766 Palacios JM, Niehoff DL, Kuhar MJ (1981): Receptor autora­ IiFurG,Guilloux F, Rufat P, Benavides J, Uzan A, Renanet diography with tritium-sensitive fIlm:Potential for com­ puterized densitometry. Neurosci Lett 25:101-105 (, Dubroeucq MC, Gueremy C (1983): Peripheral ben­ zodiazepinebinding sites: Effectsof PK 11195 1-(2 chlo­ Papadopoulos V, Mukhin AG, Costa E, Krueger KE (1990): rophenyl)-N-methyl-(1-methylpropyl)-3 isoquinolinecar­ The peripheral-type benzodiazepine receptor is function­ boxarnide. Life Sci 32:1849-1856 ally linked to Leydig cell steroidogenesis. ] BioI Chern 265:3772-3779 \iiIowitzM R, Fryer A] , Gorman]M, Campeas R, Levin A, DaviesSR, Goetz D, Klein DF (1986): Alprazolam in the Pitts W, Fann W, Sajadi e, Snyder S (1983): Open-label study treatment of panic disorders. J Clin Psychopharmacol of alprazolam in older depressed inpatients. J Clin Psy­ 6:13-20 chiatry 44:213-215 I9PI AS, Coupet ], Greenblatt EN, Klepner CA, Beer B Quirion R (1984): High density of 3H-R05-4864 «periph­ (1979):A synthetic non-benzodiazepine ligand for ben­ eral>> binding sites in the pineal gland. Eur J Pharmacol mdiazepinerec eptors: A probe for investigating neuronal 102:559-560 substrates of anxiety. Pharmacol Biochem Behav 11: Rickels K, Feighner]F, Smith WT (1985): Double-blind safety 99-106 and effIcacy study comparing alprazolam, amitriptyline, IildensH, Wisden W (1991): Function and pharmacology doxepin and placebo in the treatment of depression. Arch ofmultiple GABAA receptor subunits. Trends Phar­ Gen Psychiatry 42:134-141 macol Sci 12:49-51 Schoemaker H, Boles RG, Horst WD, Yamamura HI (1983): IIIIottiM, Mele L, De Luca C (1990): Involvement of the Specific high affInity binding sites for [3HJ-Ro 5-4864 in peripheral" benzodiazepine receptor type (C03) in the rat brain and kidney. J Pharmacol Exp Ther 225:61-69 Iolerance to the electroencephalographic effects of ben­ Sethy VH(1983): Interaction of triazolobenzodiazepineswith zodiazepines in rats: Comparison of diazepam and benzodiazepine receptors. J Pharm PharmacoI35:524-526 donazepam. Pharmacol Biochem Behav 35:933-936 Sethy VH, Harris DW (1982a): Determination of biological lUbeRT, Wamsley ]K (1986): Autoradiographic localiza­ activity of alprazolam, triazolam and their metabolites. IMln of subcomponents of the macromolecular GABA J Pharm Pharmacol 34: 115-116 receptorco mplex. Life Sci 39:1937-1945 ffUbeRT, Olsen RW, Yezuita]F, Wamsley]K (1988): Os­ Sethy VH, Harris DW (1982b): Benzodiazepine receptor num­ moticshock: A method to eliminateendogenous y-arnino­ ber after acute administration of alprazolam and diaze­ butyricacid and account for the influence on benzodi­ pam. Res Commun ChernPathol PharmacoI35:229-235 uepine binding affInity in autoradiographic studies. J Sethy VH, Hodges DH (1982): Role of beta adrenergic recep­ Pharmacal Exp Ther 245:342-349 tors in the antidepressant activity of alprazolam. Res IlUbeRT,Mahan DR, Smith RB, Wamsley]K (1990): Char­ Commun Chern Pathol Pharmacol 36:329-332 .:Ierization of [3HJalprazolam binding to central ben- Shehi M, Patterson WM (1984): Treatment of panic attacks 314 J.K. Wamsley et al. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NOA

with alprazolam and propanolo!. Am J Psychiatry 141: 1-N-trifluoroethylbenzodiazepines for the BZD-1 rece� 900-901 tors in the rat brain. Pharmacol Biochem Behav 23:973-918 Sieghart W, Karobath M (1980): Molecular heterogeneity of Wang JKT, Morgan JI, Spector S (1984a): Benzodiazepine! benzodiazepine receptors. Nature 286:285-287 that bind at peripheral sites inhibit cell proliferation. Pro: Soderpalm B, Engel JA (1989): 1l2-Adrenoceptor antagonists Nat! Acad Sci USA 81:349-351 potentiate the anticonflict and the rotarod impairing Wang JKT, Morgan JI, Spector S (1984b): Differentiationol effectsof benzodiazepines. J Neural Transm 76:191-204 friend erythroleukemia cells induced by benzodiazepine to their peripheral type-sites. Mol Pharmacol 25:349-351 Squires RF(1983): Benzodiazepine receptor multiplicity. Neu­ ropharmacology 22:1443-1450 Williams M, Olsen RW (1989): Benzodiazepine receptors and Starosta-Rubinstein S, Ciliax BJ, Penney JB, McKeever P, tissue function. In Williams M, Glennon RA, Timmer· Young AG (1987): Imaging of a glioma using peripheral mans PBMWM (eds), Receptor Pharmacology and Func· benzodiazepine receptor ligands. Proc Natl Acad Sci USA tion. New York, Marcel Dekker, pp 385-413 84:891-895 Yezuita JP, McCabe RT, Barnett A, Iorio LC, WamsleyJK UnnerstallJR, KuharMJ, NiehoffDL, Palacios JM(1981): Ben­ (1988): Use of the selective benzodiazepine-1 (BZ-1) ligand zodiazepine receptors are coupled to a subpopulation of [3H]2-oxoquazepam (SCH 15-725) to localize BZ-lrec� GABA receptors: Evidence froma quantitative autoradio­ tors in the rat brain. Neurosci Lett 88:86-92 graphic study. J Pharmacol Exp Ther 218:797-804 Young WS III, Kuhar MJ (1979): Autoradiographic localiza. Verma A, Nye JS, Snyder SH (1987): Porphyrins are endoge­ tion of benzodiazepine receptors in the brains ofhumaru nous ligands for the mitochondrial (peripheral-type) ben­ and animals. Nature 280:393-394 zodiazepine receptors. Proc Nat! Acad Sci USA 84:2256-2260 Young WS III, Kuhar MJ (1980): Radiohistochemicallocaliza. Wamsley JK, Palacios JM (1984): Amino acid and benzodi­ tion of benzodiazepine receptors in rat brain. J Pharmacd azepine receptors. In Bjorklund A, Hokfelt T, Kuhar MJ Exp Ther 212:337-346 (eds), Handbook of Chemical Neuroanatomy, Vol 2. Am­ Young WSIII, Niehoff DL, KuharMJ, Beer B, Lippa AS (1981): sterdam, Elsevier/North Holland, pp 352-385 Multiple benzodiazepine receptor localizationby lightmi­ Wamsley JK, Golden IS, Yamamura HI, BarnettA (1985): Au­ croscopic radiohistochemistry. J Pharmacol Exp Ther toradiographic demonstration of the selectivity of two 216:425-430