NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.1 57

Effect of Chronic Administration of Antidepressants on (12-Adrenoceptors in the Locus Coeruleus and Its Projection Fields in Rat Brain Determined by Quantitative Autoradiography Gyula B. Kovachich, Ph.D., Alan Frazer, Ph.D., and earl E. Aronson, Ph.D.

Thedensity of a2-adrenoceptors, using 3H-idazoxan as caused a modest increase in binding only in one terminal theradioligand, was determined by quantitative field area, whereas sertraline had no effect. Although lUtoradiography in the locus coeruleus and in 13 these antidepressants did not produce consistent effects on noradrenergic projection fields following chronic a2-adrenoceptors, protriptyline, mianserin, and Idministration of drugs acting on noradrenergic and/or phenelzine were similar in that they all decreased the StTOtonergic neurons. Protriptyline, an inhibitor of the binding of 3H-idazoxan in the locus coeruleus without JlPfake of norepinephrine, and mianserin, an widely affecting its binding in the coerulean terminal al'lUirenoceptor antagonist, reduced the binding of fields. Deprenyl, a selective inhibitor of type B MAO, the lH·idazoxan only in the locus coeruleus. Phenelzine, an only drug in this study without proven antidepressant inhibitor of both type A and type B monoamine oxidase efficacy, differedfrom all other drugs in that it decreased (MAO), reduced the binding of 3H-idazoxan in the locus the binding of 3H-idazoxan both in the locus coeruleus as CM'IIleus and in several areas with noradrenergic well as in most terminal fields with primarily coerulean innervationfrom tegmental cell bodies. Clorgyline, a noradrenergic innervation. [NeuTopsychopharmacology stltctive inhibitor of type A MAO, had no effect. Of the 8:57-65, 1993J trroselective inhibitors of serotonin uptake, citalopram

lEY WORDS: a2-adrenoceptors; 3H-Idazoxan; Locus When given repeatedly, antidepressant drugs produce uxruleus: Antidepressants multiple effects on noradrenergic and serotonergic neu­ rons and receptors (see Heninger and Charney 1987; Frazer et al. 1988). Recent autoradiographic studies identified specificregions of the brain where effectson l3-adrenergic receptors by these drugs are most promi­ From the Department of Veterans Affairs Medical Center, �ent of Psychiatry, University of Pennsylvania School of nent (Ordway et al. 1991). There has been considerable Medicine, and Laboratories of Pharmacology and Toxicology, interest in the response of a-adrenoceptors to chronic University of Pennsylvania School of Veterinary Medicine, administration of antidepressants. Some a2-adreno­ Pbiladelphia, Pennsylvania. Address reprint requests to: Gyula B. Kovachich, Ph.D., ceptors in the brain are situated in the projection ftelds t!europsychopharmacology Unit (151E), Department of Veterans of noradrenergic neurons, where they modulate the re­ Mairs Medical Center, University and Woodland Avenues, lease of norepinephrine as presynaptic autoreceptors Pbiladelphia, Pennsylvania 19104. ReceivedApril 29, 1991; revised February 1, 1992; accepted February (Langer 1977; Starke 1981), whereas others act as post­ )),1992. synaptic receptors (U'Prichard et al.1977). A number

CI993 American College of Neuropsychopharmacology Published by Elsevier Science Publishing Co., Inc. 655Avenue of the Americas, New York, NY 10010 0893-133X/93/$5.00 58 G.B. Kovachich et aI. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.1

of studies found a decrease in the density of these recep­ Animals treated once a day were injected between 0900 tors following chronic administration of antidepressant and 0930 hours, whereas those treated twice a day re­ drugs, but other studies found no effect (Pile 1987). ceived their injections at 0900 to 0930 and at 1600 to 1630 Alpha2-adrenoceptors are also found in the locus hours. Dose schedules were selected either because coeruleus. These somatodendritic adrenoceptors exert they caused alterations in �-adrenoceptors (Ordwayet an inhibitory effect on the fIring of the noradrenergic a1. 1991), or produced serotonergic autoreceptor sub­ neurons of the locus coeruleus (Svensson et a1. 1975; sensitivity (Blier and de Montigny 1985; Chaput et al Cedarbaum and Aghajanian 1976), which have wide­ 1986). The regimens for clorgyline and deprenyl are spread axonal projections throughout the brain. To the selective for type A and type B monoamine oxidase best of our knowledge no study has dealt with the effect (MAO), respectively Oohnstone 1968; Campbell et al. of differenttypes of antidepressant drugs on the den­ 1979; Eckstedt et al. 1979). The doses of sertraline 01 sity of o.2-adrenoceptors in this cell body area. How­ protriptyline have been shown to block in vivo the up­ ever, a number of studies have dealt with the effectof take of serotonin (5-HT) (Wolfe et al. 1987) or norepi· repeated administrationof antidepressant drugs on the nephrine (Schildkraut et al. 1971), respectively. spontaneous and evoked electrical activity of the locus coeruleus (Scuvee-Moreau and Svensson 1982; Blier Preparation of Tissue Sections and de Montigny 1985; Campbell et al. 1985; Curtis and Valentino 1991). These studies showed that whereas Rats were euthanized by decapitation between 1000and some antidepressants do cause changes in locus 1100 hours the morning after receiving their last injec· coeruleus fIring or the response of these cells to 0.2- tion of drug or saline (i.e., 18 to 25 hours after the £mal adrenoceptor agonists, others do not produce such injection). The brains were removed, rinsed in ice-cold effects. saline, and frozen on powdered dry ice. The frozen tis· The present study examined the effectof repeated sue was stored at -80aC until sectioning. From each administration of antidepressant drugs on the binding brain, two sets of coronal sections were cut 20 J.Lm in of 3H-idazoxan, a highly selective o.2-adrenoceptoran­ thickness in a cryostat at -15aC. One set of sections tagonist (Chapleo et al. 1981; Dettmar et al. 1983), to was cut at the level of the dorsomedial hypothalamic o.2-adrenoceptors in the locus coeruleus and in the ter­ nucleus and the other was cut in the mid-level of the minal fIelds of coerulean neurons. The measurements locus coeruleus. The sections were thaw mounted onto were obtained using the technique of quantitative recep­ gelatin-coated frozen microscope slides and dehydrated tor autoradiography, the best available method for overnight at 4°C. Several sections from each serially measuring receptors in discrete regions of the brain (Ku­ cut set were stained with thionin and examined under har 1985). a light microscope to establish their position along the rostral-caudal axis, using the stereotaxic atlas by Pax· inos and Watson (1986) as reference. With the aid of MATERIALS AND METHODS these stained sections the rostral and caudal limits of Figure 29 of the atlas were identifIed.By a similar pro­ Animals cess, the rostral and caudal limits of Figure 58 of the Male Sprague-Dawley rats (weighing between 200 and atlas, showing the most densely packed cells of the lo­ 225g) were purchased from Ace Animals (Boyertown, cus coeruleus, were bracketed. Two to four sections at PA) and housed in clear polycarbonate cages in groups each level were incubated. that did not exceed four animals per cage. The rats re­ ceived Purina Rodent Lab Chow #5001 (Purina Corp., Incubation Procedure St. Louis, Missouri) and tap water ad libitum. Light in the animal quarters was regulated on a 12-hour In preparation for incubation, the sections were trans· light/dark cycle with the room being dark from 1800 ferred from a freezer at -80aC to a desiccator at 4°( hours until 0600 hours. for temperature equilibration.The incubation procedure was performed essentially as described by Boyajian et a1. (1987) and Chamba et al. (1991). First, the sections Administration of Drugs were preincubated at room temperature in buffer (58 Table 1 shows the dose, route, and schedule of adminis­ mmollL Na2HP04, 8.5 mmollL KH2P04, pH 7.4) for 15 tration for each drug used in this study. The drugs, minutes. Incubation was carried out at 4°C for 2 hours whose concentrations are expressed as the free base, in buffer containing 3.0 nmollL 3H-idazoxan (42-53 were dissolved in distilled water so as to permit injec­ Ci/mmol, Amersham Corp., Arlington Heights, IL).A tion of a volume of 0.1 ml/l00 g of body weight for all maximum of fIveslides, containing two sections each, drugs except for mianserin and sertraline, which were were placed in 30 ml of incubation medium in polyeth· injected in a volume of 0.2 ml/l00 g of body weight. ylene Coplin jars. Upon completion of incubation, the NEUROPSYCHOPHARMACOLOGY1993-VOL. B, NO. 1 3H-Idazoxan Binding and Antidepressants 59

Table 1. Treatment Regimen For Orugsa

Drug Dose Route No. Administrations No. Rats

Saline 1 ml/kg IF once daily 14 Citalopram 20 mg/kg IF once daily 5 Clorgyline 1 mg/kg SC once daily 7 Oeprenyl 0.25 mg/kg SC once daily 5 Mianserin 15 mg/kg IF twice daily 7 Phenelzine 5 mg/kg IF once daily 8 Protriptyline 10 mg/kg IF twice daily 6 Sertraline 5 mg/kg IF twice daily 5

a Treatments were for 21 days, except for citaiopram, which was administered for 14 days. sectionswere washed twice for 1 minute in 50 mmollL squares parametric curve fItting program (Sigmaplot Trisbuff er, pH 7.6, at 4°C, dipped into deionized wa­ 4.0, J andel ScientifIc, CorteMadera, CA). One-site and ter at room temperature, and then air-dried on a slide two-site models were compared using a Fisher ratio (F) warmer. NonspecifIc binding was defIned using 10 computed as follows: IlmollLphentolamine as a displacing agent, based on F = (551 - SS2)/dfss1 - sS2/SS2ldfss2, theresults of displacement experiments (see Results). NonspecifIc binding was approximately 15% of total where 551 and 552 are the error sum of squares for the binding in the locus coeruleus and 20% to 25% in one-site model and two-site model, respectively; dfss1 noradrenergic terminal fIelds. Total binding and non­ and dfss2 are the degrees of freedom for the one-site specifIcbinding were determined on adjacent sections. and two-site models. The two-site model is accepted as a signifIcantlybetter fItthan the one-site model when the value of F is greater than or equal to the tabulated Quantitative Autoradiography value at the given degrees of freedom and the proba­ The dried, slide mounted sections were placed into bility of a type I error of 0.05. spring-loaded metal x-ray cassettes along with plastic embeddedtritium standards (American Radiolabelled Drugs Chemicals, St. Louis,MO), previously calibrated against brain mash standards according to the method de­ The antidepressants used in this study were citalopram scribed earlier (Geary and Wooten 1983; Geary et al. (H. Lundbeck, Copenhagen, Denmark), clorgyline 1985), andapposed at room temperature to tritium sen­ (May and Baker Ltd., London, England), deprenyl sitive &lm (Ultro&lm 3H, Cambridge Instruments, (Somerset Pharmaceutical, Inc., Denville, N.J.), mianse­ Gaithersburg, MD). Sections containing the locus rin (Organon, Int., Oss, The Netherlands), phenelzine coeruleus were exposed for 28 days and those show­ (Sigma Chemical Co., St. Louis, MO), protriptyline ingterminalfIeld areas were exposed for 49 days. The (Merck, Sharp and Dohme Research Labs, West Point, exposed fIlms were processed using Kodak GBX de­ PA), and sertraline (PfIzer Inc., Groton, CT). veloper and fIxer. The autoradiograms were analyzed on a DUMAS (Drexel Unix based Microcomputer Im­ age Analysis System) densitometer using the BRAIN RESULTS softwarepackage (Feingold et al. 1986). The incubated sections were subsequently stained with thionin to The radioligand 3H-idazoxan is a highly selective a2- facilitateidentiftcation of neuroanatomic structures that adrenoceptor antagonist (Chapleo et al. 1981; Dettmar were defined and named according to the atlas of Pax­ et al. 1983); however, it binds to sites other than a2- inos and Watson (1986). adrenoceptors and a portion of this non-a2 adrenocep­ tor binding is displacable by certain a2-adrenoceptor antagonists (Michel et al. 1989; Brown et al. 1990; Wik­ Statistical Methods berg and Uhlen 1990). Consequently, we carried out Thesamples were analyzed blind and identiftedby ran­ experiments to ascertain if3H-idazoxan was binding to domly chosen code numbers. The data were examined non-a2-adrenoceptors under our experimental condi­ byanal ysis of variance (Sokal and Rohlf 1969), and in­ tions. Experiments were also carried out to determine dividual groups were compared to the control group what nonradioactive drug would best defIne the bind­ byDunne tt's test (Zar 1984). P values of less than 0.05 ing of 3H-idazoxan to a2-adrenoceptors, i.e., which were considered signifIcant. In competition experi­ drug should be used to defIne specifIc binding. ments the data were analyzed using a nonlinear least- Competition experiments were carried out to ascer- 60 G.B. Kovachich et al. NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO.1

tain the extent of displacement of total binding of 3H_ VI :::J a W 100 a idazoxan. In these experiments coronal sections were ---' :::J used either from the level of the midbrain, where the et:: W terminal fIelds were analyzed, or from the level of the a u 80 locus coeruleus; binding of 3H-idazoxan was measured VI :::J either by total radioactivity per section determined by U liquid scintillation spectroscopy or by quantitative au­ a ---' GO toradiographic analysis of the locus coeruleus, respec­ C> tively. The concentration of 3H-idazoxan used was 3 Z nmollL, the same as that in the subsequent experi­ 0 Z 40 ments. Figure 1A shows the results of competition CD ---' experiments with the adrenergic antagonists phentola­ < f- mine, prazosin, SKF-104375, and the physiologic neu­ a 20 f- rotransmitter ( - )norepinephrine in the locus coeruleus. LL These drugs were selected as they do differentiate a2- a 1lI1 adrenergic from non-a2-adrenergic binding sites of 3H_ 0 -1 0 J 5 idazoxan (Michel et al. 1989). It is evident from the il­ LOG CONCENTRATION (nM) lustration that each drug displaced approximately 80% to 90% of total binding of 3H-idazoxan. Nonlinear regression analysis indicated that the displacement 2500 curves were adequately fIt by a one-site model for all of the drugs. The Ki values were in agreement with � W those obtained by Boyajian and Leslie (1987). Similar f- T 0 2000 results were obtained when samples from the midbrain et:: n. were incubated and radioactivity per section was de­ C> termined by liquid scintillation spectroscopy (results not ::::!! "- 15DO 0 NS shown). From such data, it was inferred that only a Z :::J small portion of the total binding of 3H-idazoxan was a CD to non-a2-adrenoceptors. Further, these results indi­ 1000 Vl cated that 10 IlmollL phentolamine could be used to UJ ---' s defIne the specifIc binding of 3H-idazoxan to a2- a :::;; a 500 adrenoceptors. f- Z To characterize further the binding of 3H-idazoxan, UJ LL saturation experiments were carried out. In these ex­ periments randomized coronal sections were used from 0 0 2 4 • 8 the level of the caudate-putamen and total radioactiv­ JH-IDAZOXAN (nM) ity of sections was determined by liquid scintillation spectroscopy. The results, shown in Figure lB, indi­ Figure 1 A: Competition with binding of 3H-idazoxan cate that 3H-idazoxan bound to a single class of bind­ (3nmollL) in the locus coeruleus by adrenergic antagonists ing sites with a Kd of 0.99 ± 0.23 nmollL (mean ± SD, and ( - )norepinephrine. The drugs used are phentolamine (PH), prazosin (PR), (SKF), and (- )norepine­ n = 3), in agreement with the value reported by SKF-I04078 Boyajian et al. (1987). Insubsequen t experiments, a con­ phrine (NE). The curves for PH, PR, and NE represent the mean of three experiments, the curve for SKF is the mean centration of 3 nmollL 3H-idazoxan was used, three of fIve experiments. B: Saturation isotherm for 3H-idazoxan. times its Kcl value. This concentration of 3H-idazoxan Randomized coronal sections were used from the level of the occupies 75% of a2-adrenoceptors, which increases the caudate-putamen. T = total binding; S = specifIc binding likelihood that a recorded change in the binding of 3H_ NS = nonspecifIcbinding. Radioactivity per section was de­ idazoxan reflects an alteration in the density of bind­ termined by liquid scintillation counting. NonspecifIcbind· ing sites, rather than a change in the affinityof the bind­ ing was defIned by 10 IJ.mollL phentolamine. This fIgure ing site for the ligand. shows one of three experiments. The effectof chronic administration of drugs on the binding of 3H-idazoxan in the locus coeruleus is shown The effectof chronic administration of these drugs in Figure 2. Four of the drugs, namely protriptyline, on the binding of 3H-idazoxan in terminal fIeld areas mianserin, phenelzine, and deprenyl, caused modest is summarized in Table 2. In these structures, only (13% to 18%) but signifIcant (p < 0.01) decreases in the deprenyl and phenelzine altered binding signifIcantly. binding of 3H-idazoxan in the locus coeruleus. Treat­ Both drugs reduced the binding of 3H-idazoxan in the ment with clorgyline, sertraline, and citalopram had no basomedial amygdaloid nucleus to approximately the effect. same degree (about 13%). Phenelzine also reduced NEUROPSYCHOPHARMACOLOGY 1993-VOL. 8, NO. 1 3H-Idazoxan Binding and Antidepressants 61

cus coeruleus and in some terminal neld areas, mianse­ rin and protriptyline had no effect on the binding of this radioligand in projection nelds (Table 2). Finally, citalopram produced a modest (8%), but statistically signiftcant increase in binding of 3H-idazoxan in the dorsal hypothalamic area, whereas sertraline and clor­ gyline had no statistically signiftcanteffect in any of the terminal nelds examined (results not shown).

DISCUSSION figure 2 The effect of antidepressants (3 nmoliL on the The radioligand 3H-idazoxan, with 10 IlmollL phen­ speci.fJc binding of 3H-idazoxan in the locus coeruleus. Data tolamine as the displacing agent to denne speciftc bind­ mean SD; n = 14 for control samples and 6-8 shown are ± ing, has been used for quantitative autoradiographic for treated samples. * = p < 0.01. measurement of az-adrenoceptors prior to this study (Boyajian et al. 1987; Boyajian and Leslie 1987; Chamba binding signiftcantly in the dorsal hypothalamic area et al. 1991). Others speculated that, due to the pres­ (18%), in the central amygdaloid nucleus (16%), and ence of a nonstereoselective adrenergic binding site, the inthe paraventricu1arthalamic nucleus (13%). Deprenyl density of az-adrenoceptors may be overestimated by reduced binding in all the cortical areas analyzed. The this method (Convents et al. 1989; Lehmann et al. 1989). greatest effects were produced in Areas 1 and 2 of the This speculation was based on measurements per­ frontalco rtex (22%), in the hindlimb area of the cortex formed on homogenates of human and rabbit cortex (18%), and in the retrosplenial cortex (17%). More mod­ (Convents et al. 1989) in the presence of a buffer con­ est reductions in binding were found in the parietal cor­ siderably different and at a concentration of 3H-ida­ tex, in Layers 2 through 6 (12%). Layer 1 was not ana­ zoxan signiftcantly higher than that used in the au­ lyzedbecause this narrowsuperftci al structure oftendid toradiographic procedure. The displacement curve not remain intact. produced by phentolamine under those conditions was In contrast to the ability of these inhibitors of MAO shallow and biphasic, and 10 IlmollLphentolamine only to decrease the binding of 3H-idazoxan both in the 10- displaced approximately 40% of the total binding. This

Table 2. The Effect of Antidepressants on the SpecifIc Binding of 3H-Idazoxan (3 nmollL) in Noradrenergic Terminal Field Areas·

Noradrenergic Innervation Primarily Coerulean

Area Saline Protripb Mianserin Phenelzine Deprenyl

Hindlimb area of cortex 349 ± 5 339 ± 6 365 ± 5 341 ± 9 288 ± 8d Retrosplenial Cortex 399 ± 10 373 ± 9 400 ± 12 363 ± 12 331 ± 16d Frontal Cortex Area 1 & 2 354 ± 9 340 ± 16 366 ± 15 334 ± 19 275 ± 18d Parietal Cortex Layers 2, 3, 4 455 ± 10 412 ± 23 470 ± 8 416 ± 11 401 ± 18c Parietal Cortex Layer 5 356 ± 10 330 ± 14 357 ± 13 324 ± 8 312 ± 15c Parietal Cortex Layer 6 325 ± 10 305 ± 18 331 ± 12 298 ± 8 285 ± 14C Basomedial Amygd. N. 1129 ± 30 1067 ± 64 1114 ± 35 975 ± 54c 986 ± 26c Laterodorsal Thalamic N. 784 ± 37 724 ± 73 897 ± 14 797 ± 35 863 ± 46

Noradrenergic Innervation Primarily Noncoerulean

Area Saline Protripb Mianserin Phenelzine Deprenyl

Paraventr. Thalamic N. 943 ± 16 919 ± 43 1003 ± 31 819 ± 25c 886 ± 31 Ventromed. Hypoth. N. 741 ± 21 667 ± 92 772 ± 31 676 ± 25 655 ± 39 Lateral Hypoth. Area 657 ± 17 549 ± 73 643 ± 37 586 ± 23 604 ± 40 Dorsal Hypoth. Area 927 ± 12 894 ± 25 933 ± 35 759 ± 27c 860 ± 31 Central Amygd. N. 1022 ± 41 955 ± 35 1011 ± 29 855 ± 22C 946 ± 55

• Data are shown as mean ± SEM (fmole/mg protein); n = 14 for control and 5-8 for treated samples. b protriptyline. c p < 0.05. d P < 0.01. 62 G.B. Kovachich et aI. NEUROPSYCHOPHARMACOLOGY199 3-VOL. 8, NO.1

result ismarkedly different from what we observed un­ not an effectiveantidepressant medication (Mendlewicz der our assay conditions, using slide-mounted sections and Youdim 1983; Murphy et al. 1987). The other an· of rat brain and 3 nmol/L 3H-idazoxan. Both ( - )nore­ tidepressants, namely clorgyline, an inhibitor of type pinephrine, the physiologically active neurotransmit­ A MAO Oohnston 1968; Hall et al. 1969), sertraline and ter, and phentolamine produced steep monophasic dis­ citalopram, selective inhibitors in vivo of the uptake � placement curves and displaced 3H-idazoxan to the 5-HT(Koe et al. 1983; Hyttel and Larsen 1985), did not same extent: approximately 80% of the total binding have this effect. in the locus coeruleus (Fig. lA), and 70% to 80% of the Antidepressant drugs have dissimilareffects on the total binding in the terminal fIelds (data not shown). fIring activity of the locus coeruleus. For example, the Also, 3H-idazoxan binds to a nonadrenergic (idazoxan) spontaneous fIring of the locus coeruleus shows a sus­ receptor, in addition to u2-adrenoceptors, and in some tained decrease during chronic administration of either tissues as much as 50% of the total binding by 3H-ida­ clorgyline or phenelzine (Blier and de Montigny 1985), zoxan is to this nonadrenergic site (Michel et al. 1989; but it is not affected by repeated administration of mi· Brown et al. 1990; Wikberg and Uhlen 1990). Certain anserin (Curtis and Valentino 1991). On the other han4 adrenergic antagonists, such as phentolamine and acute administration of imipramine and desipramine prazosin, displace 3H-idazoxan only from u2-adreno­ markedly inhibits the fIringof the locus coeruleus, but ceptors, whereas others, such as SKF-I04078and oxy­ during repeated administration of these drugs theirin· metazoline, are capable of displacing 3H-idazoxan from hibitory effect on the electrical activity of the locus both the u2-adrenoceptors as well as from the non­ coeruleus becomes less pronounced (Svensson and Us­ adrenergic (idazoxan) receptor (Michel et al. 1989). Fig­ din 1978; McMillen et al. 1980; Scuvee-Moreau and ure lA shows that in the locus coeruleus the binding Svensson 1982). It was proposed that the gradual dis­ of 3H-idazoxan is displaced by SKF-I04078to a slightly sipation of the inhibitory effect of imipramine and greater extent than by either phentolamine or prazo­ desipramine was due to the development of subsensi· sin. Similar results were obtained in the terminal fIelds tive u2-adrenoceptor responsiveness, since microion· (results not shown). These results indicate that although tophoretically applied clonidine, an u2-adrenoceptOl a minor portion (perhaps 10%) of the total binding by agonist, produced a diminished inhibitory effecton the 3H-idazoxan may be to the idazoxan receptor in our fIring of the locus coeruleus after continued adminis samples, nevertheless, phentolamine does not recog­ tration of those drugs (McMillen et al. 1980). nize this site, in agreement with the fIndingsof Michel Our autoradiographic data are consistent with the et al. (1989). Taken together, these fIndingsdemonstrate electrophysiologic measurements in that chronic ad· that our assay conditions, using 3H-idazoxan and 10 ministration of protriptyline, which like imipramineand IJ.mol/Lphentolamine to defInespecifIc binding, are ap­ desipramine inhibits the uptake of norepinephrine, de­ propriate for measurement of u2-adrenoceptors in the creased the density of u2-adrenoceptors in the locus locus coeruleus and in the terminal fIelds at the level coeruleus. Furthermore, chronic administration of elOl' of the midbrain using slide-mounted sections. gylirie had no effect on either the sensitivity of locus Chronic administration of some of the antidepres­ coeruleus cells to clonidine (Blier and de Montigny 1981; sant drugs studied decreased the density of u2-adreno­ Campbell et al. 1985) or on the binding of 3H-idazoxan ceptors in the locus coeruleus. These drugs were pro­ in the locus coeruleus. On the other hand, whereas we triptyline, an inhibitor of the uptake of norepinephrine found a modest decrease in the density of u2-adreno­ (Schildkrautet al. 1971; Richelson and Pfenning 1984), ceptors in the locus coeruleus after repeated adminis­ phenelzine, an inhibitor of both type A and type B MAO tration of phenelzine or deprenyl, similar treatmenb (Robinson et al. 1979), and mianserin, an u2-adreno­ produced no changes in the response of the locus ceptor antagonist (Baumann and Maitre 1977; Engberg coeruleus to clonidine (Blier and de Montigny 1985) and Svensson 1980). It is unlikely that the observed de­ The daily dose of phenelzine in the latter study, how­ crease in binding after treatment with mianserin is due ever, was considerably lower than that used in our ex· to the presence of residual drug. Allsamples were prein­ periments. Both the electrophysiologic and the au­ cubated for 15 minutes (350 ml buffer per 25 sections) toradiographic measurements indicate, nevertheless prior to incubation with 3H-idazoxan; some samples that the effects by different types of antidepressant from mianserin-treated rats were preincubated for 45 drugs on the responsivity or density of u2-adreno­ minutes and they produced results similar to those that ceptors in the locus coeruleus are not uniform. were preincubated for only 15 minutes. Deprenyl, an Only two of the drugs, deprenyl and phenelzine inhibitor of type B MAO (Knoll and Magyar 1972), a produced substantial reductions in binding of 3H drug best known for potentiating L-DOPA treatment idazoxaJ)in noradrenergic terminal fIelds. Some of tm in parkinsonian patients (Birkmayer et al. 1983), also terminal fIeldsexamined in this study receive all of th� reduced the binding of 3H-idazoxan. Although the noradrenergic fIbers from the locus coeruleus, namelr' data are not yet conclusive, it appears that deprenyl is the parietal cortex, frontal cortex, retrosplenial cortex NEUROPSYCHOPHARMACOLOGY 1 993 - VOL. 8, NO. 1 3H-Idazoxan Binding and Antidepressants 63

hindlimbarea of the cortex, laterodorsal thalamic nu­ outnumber the presynaptic ones (Greenberg et al. 1976; cleus, and basomedial amygdaloid nucleus. Other brain V'Prichard et al. 1977), it is likely that our results with areasreceive either mixed coerulean and noncoerulean 3H-idazoxan in terminal fIeld areas represent binding (i.e., tegmental) noradrenergic innervation, or receive predominantly to postsynaptic a2-adrenoceptors. The only noncoerulean noradrenergic innervation, namely a2-adrenoceptor population has been divided into at the paraventricular thalamic nucleus, ventromedial least two subtypes, a2A- and a2s-adrenoceptors, based hypothalamic nucleus, dorsal hypothalamic area, lateral on their pharmacologic properties (Bylund et al. 1988). hypothalamic area, and central amygdaloid nucleus Our competition experiments, involving the locus (Lindvall and Bjorklund 1978; Moore and Card 1984; coeruleus (Fig. 1A) and terminal fIeldsat the level cor­ Aston-Jones et al. 1984). responding to Figure 29 of the atlas by Paxinos and Wat­ Deprenyl, an inhibitor of type B MAO, produced son (1986), indicate that 3H-idazoxan binds to a single a distinctive pattern of effect in the nor adrenergic ter­ population of a2-adrenoceptors. Further studies are minalfIelds (Table 2): it reduced the binding of 3H-ida­ needed to determine whether these adrenoceptors are zoxan in seven of the eight areas of the brain with of the a2A or a2B subtypes. noradrenergicprojections originating only from the lo­ In conclusion, whereas some antidepressants in this cus coeruleus, whereas its effects were consistently study showed no effect, others produced modest smaller and were statistically not signifIcant in brain decreases in a2-adrenoceptors in distinctive regional areasthat receive noradrenergic innervation, either ex­ patterns. Further studies are needed to examine the clusively or partially, from tegmental cell bodies. signifIcanceof these fIndingswith regard to the mech­ Phenelzine, an inhibitor of type A and type B MAO, anism of action of antidepressant drugs. decreased the binding of 3H-idazoxan primarily in those terminalfIeld areas where deprenyl had no effect, namely those that receive noradrenergic projections, ACKNOWLEDGMENTS ei therexclu sively or partially, from tegmental cell bod­ ies(Table 2). Given this, together with the fact that clor­ This work was supported by research funds from the Depart­ gyline, a type A MAO, had no effect, it is not clear how ment of Veterans Affairs and U.s. Public Health ServiceGrant MH29094. theeffects ona2-ad renoceptors by deprenyl or phenel­ zine are related to inhibition of MAO. Interestingly, phenelzine produced similar regional REFERENCES effectson �2-, but not on 131-adrenoceptors (Ordway et aI. 1991), as on a2-adrenoceptors. 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