Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and ␣ 2-Adrenergic Receptors in Human Brain John E. Piletz, Ph.D., Gregory A. Ordway, Ph.D., He Zhu, M.D., Betty J. Duncan, B.S., and Angelos Halaris , M.D., Ph.D.

␣ Clonidine is a partial agonist at brain 2-adrenoceptors not enriched in I-sites. Competition curves were generated ␣ ϭ ( 2AR), but also has high affinity (KD 51 nM) in for I-sites in caudate sections using 10 ligands known to homogenate binding assays for non-adrenergic imidazoline- distinguish between I1 and I2 subtypes. The rank-order of binding sites (I-sites; imidazoline receptors). Herein, an affinities were cirazoline Ͼ harmane Ͼ BDF6143 Ͼ autoradiographic comparison of [3H]-clonidine binding to idazoxan ϭ tizanidine (affinities of agmatine, efaroxan, ␣ I-sites and 2AR in sections of human brain is reported. For moxonidine, NE, and oxymetazoline were too low to be I-sites, the adrenergic component of 50 nM [3H]-clonidine reliable). Only the endogenous I-site ligand, harmane, had a binding was masked with either 60 ␮M norepinephrine monophasic displacement curve at the non-adrenergic sites ␣ ␮ ϭ Ϯ (NE; 2AR agonist) or 12.5 M methoxy-idazoxan (Ki 521 12 nM). In conclusion: 1) the distribution of ␣ 3 (MIDX; selective 2AR antagonist), whereas the remaining non-adrenergic [ H]-clonidine binding sites in human brain ␣ non-adrenergic sites were studied by displacement with 20 sections was correlated with, but distinct from 2AR; and ␮ 3 ␣ ␣ ␣ M cirazoline. Levels of [ H]-clonidine binding to 2AR 2) the affinities of these sites was distinct from 1AR, 2AR, and I-sites, determined in adjacent tissue sections, were I1 or I2 sites as previously defined in membrane binding positively correlated across 27 brain regions (p ϭ 0.0003; assays. The properties of this non-adrenergic [3H]-clonidine r2 ϭ 0.385). The principal olivary nucleus and the rostral binding site are consistent with I-sites previously labeled by portion of the ventrolateral medulla had highest ratios of [3H]-cirazoline in rat brain. ␣ Ͼ I-sites: 2AR ( 4:1). Quantitative transepts drawn across [Neuropsychopharmacology 23:697–708, 2000] ␣ hippocampal images revealed 2AR enrichments in the CA- © 2000 American College of Neuropsychopharmacology. 1 and inner molecular layers of the dentate gyrus—areas Published by Elsevier Science Inc.

␣ KEY WORDS: Imidazoline receptors; 2-Adrenoceptors; Clonidine is the prototypic imidazoline compound that Clonidine; Cirazoline; Hippocampus; Moxonidine can elicit a diversity of CNS effects (Buccafusco 1992). ␣ Originally thought to be a selective agonist for 2- ␣ From the Departments of Psychiatry and Human Behavior (JEP, adrenoceptors ( 2AR), clonidine is now realized to pos- GAO, HZ, BJD, AH), Pharmacology and Toxicology (JEP, GAO, sesses only slightly lower affinity for non-adrenergic AH), and Physiology and Biophysics (JEP), University of Missis- imidazoline receptors (IR) compared to ␣ AR (Bousquet sippi Medical Center, Jackson, MS. 2 Address correspondence to: John E. Piletz, Ph.D., Department of et al. 1984; Ernsberger et al. 1987). Psychiatry, University of Mississippi Medical Center, Rm. G128, The first subtype of IR (IR1) was discovered by com- 2500 North State Street, Jackson, MS, 39216–4505. paring the hypotensive effects of clonidine to other Drs. J. Piletz and G.A. Ordway contributed equally to this work. ␣ Received 28 January 2000; revised 25 May 2000; accepted 31 May non-imidazoline 2AR agonists, which were far less hy- 2000. potensive agents than clonidine when micro-injected

NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 © 2000 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/00/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(00)00166-4

698 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

␣ into the rostral ventrolateral medulla (RVLM) of anes- In summary, we are describing a non- 2AR binding thetized rats (Bousquet et al. 1984). In those studies, the site for [3H]-clonidine which has a distribution distinct 3 ␣ pharmacological control of blood pressure was found from [ H]-clonidine-labeled 2AR in human brain, and to best correlate with agents classified as imidazoline the pharmacology of this site resembles an imidazoline compounds (Ernsberger and Haxhiu 1997). In fact, the binding site previously thought by De Vos and cowork- recommended therapeutic doses for clonidine and ers (1994) to be an I1 site. other imidazoline compounds to treat patients with hy- pertension have been positively correlated with their affinities for non-adrenergic imidazoline binding sites

(of the I1 subtype)—whereas no such correlation exists MATERIALS AND METHODS with their affinities for ␣ AR (Buccafusco et al. 1995). 2 Human Tissue Collection In psychiatry, novel imidazoline compounds are cur- rently being explored for possible neuro-protective Discarded brain tissues from five subjects (three males properties (Regunathan et al. 1999), for treating drug and two females) who died of sudden natural or acci- withdrawal symptoms (Vandergriff et al. 1999), and as dental causes were collected at the time of autopsy at possible antidepressants (Raddatz and Lanier 1997). the Cuyahoga County Coroner’s Office, Ohio, in accor- Second-generation imidazoline anti-hypertensives are dance with an approved Institutional Review Board also now available, which include rilmenidine and mox- protocol. Tissues were coded to protect the identity of onidine (Molderings 1997). Rilmenidine and moxoni- subjects. Deaths occurred within four hours of injury. dine possess approximately 5-fold and 100-fold selec- Causes of death were determined by the county medical ␣ tive affinities, respectively, for I1 over 2AR sites examiner. Four of the subjects died of cardiovascular (Ernsberger et al. 1995a; Piletz et al. 1996), and this affin- failure and one died of asphyxiation secondary to ity difference probably explains their diminished seda- smoke inhalation. The average age of the subjects was tive side-effects relative to clonidine (van Zwieten 1997). 52 Ϯ 10 yr (age range ϭ 24 to 80 yr). All cadavers were Like most receptor families, there are several IR sub- refrigerated prior to autopsy, immediately upon arrival types identified using radioligand binding techniques. at the coroner’s facility. The average postmortem interval Ϯ I1 sites have high affinity and I2 sites have low affinity (from death to freezing of brain tissue) was 16 3 h for clonidine (Michel and Ernsberger 1992). By contrast, (range 6 to 21 h). The coroner performed toxicological planar imidazoline compounds, like idazoxan, exhibit screens for 50 compounds in the blood, bile, urine, and/ preferential affinity for I2 sites, of which there are two or vitreous fluids of cadavers to identify numerous psy- subclasses: I2A and I2B (Eglen et al. 1998). There are at choactive and other compounds. Four of the subjects least two other I-site subtypes defined by various other had no detectable drugs in the toxicological screen. One imidazolines: a cirazoline-sensitive I-site in kidney and subject had evidence of lidocaine in the blood. Postmor- brain (Angel et al. 1995) and a novel efaroxan-sensitive tem autopsies revealed no record of neurological or I-site in pancreas (Monks et al. 1999). In addition, the psychiatric histories for any of the subjects. search for endogenous ligands has identified two puta- At autopsy, blocks of brain tissue were dissected. tive IR neurotransmitters: a decarboxylated metabolite The techniques for dissection of blocks of tissue have of arginine, agmatine (Reis and Regunathan 1999), and been published for frontal cortex (Brodmann’s area 10) a ␤-carboline, harmane (Hudson et al. 1999). and hippocampus (Klimek et al. 1999), and for locus co- Only one previous autoradiographic study exists of eruleus (Klimek et al. 1997). The tissue block containing 3 [ H]-clonidine binding to brain I1 sites (De Vos et al. the hypothalamus was dissected just anterior to the 1994). While improving on that autoradiographic method, substantia nigra and was sectioned in the coronal plane. we have now provided a more detailed visualization of A block of tissue containing the medulla was obtained those sites in human brain. A comparison is made of by a transverse cut just caudal to the caudal end of the the distribution of non-adrenergic versus adrenergic locus coeruleus, and this block of tissue was cut in the sites labeled by [3H]-clonidine across 27 brain regions. transverse plane, perpendicular to the rostro-caudal De Vos et al. (1994) did not previously distinguish non- axis of the medulla. Tissue blocks were dipped in adrenergic versus adrenergic sites labeled by [3H]- 2-methylbutane cooled on dry ice (Ϫ50ЊC), placed on clonidine. Special attention is given to the hippocam- powdered dry ice for 10 min, and then stored in an ul- pus, where previous studies of depressed suicide vic- tra-cold freezer (Ϫ82 ЊC) until sectioned. Frozen blocks tims have previously revealed a decrease in IR immu- of brain tissue were mounted on a specimen chuck of a noreactivity by western blotting (Piletz et al. 2000b). A cryostat microtome (Cryocut 1800, Leica, Reichert-Jung, pharmacological rank-ordering of affinities is also pre- Deerfield, IL) and tissue sections (20 ␮m thick) were cut sented for I-sites labeled by [3H]-clonidine using com- at Ϫ16ЊC and thaw-mounted onto gelatin-coated micro- ␣ petitive ligands known to distinguish I1, I2, and 2AR scope slides. Sections were dried under refrigeration sites (taken from previous homogenate binding studies). and stored at Ϫ82ЊC until assayed.

NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 Clonidine Binding Sites in Brain 699

surements were made on serial adjacent caudate sec- Quantitative Autoradiography tions (2–4 sections per slide, wiped together). After in- Quantitative autoradiography of [3H]-clonidine bind- cubation for 1 h and a rapid cold water dip, the caudate ing was performed using a modification of the method sections were wiped from the slides onto S&S #32 glass of De Vos et. al. (1994). [3H]-clonidine (50 nM) was used fiber filters (Schleicher and Schuell, Keene, NH). Radio- ␣ to label both 2AR and I-sites in adjacent tissue sections. activity was measured by liquid scintillation counting. Sections were removed from the freezer and dried on In a typical experiment, total radioactivity remaining in the counter top at room temperature (22ЊC). Subse- the presence of 60 ␮M NE was 1503 Ϯ 222 cpm, and quently, sections were incubated with [3H]-clonidine, in that remaining in the presence of 20 ␮M cirazoline plus the presence and absence of competitors as designated 60 ␮M NE was 433 Ϯ 28 cpm. Competition curves were below, in a reaction buffer containing 50 mM Tris-HCl, analyzed by both LIGAND (McPherson 1985) and ␮ 2 mM MgCl2, and 252 M ascorbic acid (pH 7.4). This Prism (GraphPad, San Diego, CA), producing similar was performed by placing a small drop of reaction results. Data were modeled to one-site and two-site fits buffer containing the radioligand, directly on each (McPherson 1985) and the preferred model was chosen

slide-mounted section, and then incubating 1 h in a hu- by an F test. IC50 values were computed from 2-3 exper- midity chamber at 22ЊC. Unbound radioligand was re- iments and were expressed as mean Ϯ SEM. moved by three consecutive washes (1 min each) of the slides in ice-cold reaction buffer, then dipped for 1 sec Drugs in ice cold water to remove buffer salts. Slide-mounted sections were dried immediately afterwards under a [3H]-clonidine (66.5 Ci/mmol) was purchased from stream of air at room temperature overnight. Slides NENTM (Life Science Products, Inc., Boston, MA) and were apposed, along with brain-mash calibrated 3H stored at Ϫ20ЊC before reconstitution in reaction buffer standards (American Radiolabeled Chemicals Inc., St. (defined below). Moxonidine and BDF6143 were gifts Louis, Mo.), to Hyperfilm-3H (Amersham, Arlington of Lilly Forschung GmbH Company (Hamburg, Ger- Hts, IL) in x-ray cassettes and stored at room tempera- many). Tizanidine was a gift of Sandoz Pharmaceuti- ture for four weeks. Films were processed with GBX de- cals (Tokyo, Japan). Efaroxan was a gift from Pierre veloper and fixer (Eastman Kodak Co., Rochester, NY). Fabre Pharmacie (Castres Cedex, France). Cirazoline The same sections were then stained with cresyl violet and RX821002 (MIDX; methoxy-idazoxan) were pur- to identify anatomical structures. The resulting autora- chased from Research Biochemicals International (Nat- diograms were quantified using the M2 Microcomputer ick, MA). Agmatine, harmane, NE, oxymetazoline, and Imaging Device (Imaging Research, St. Catharines, On- idazoxan (IDX) were from Sigma Chemical Company tario, Canada). Hippocampal fields (Klimek et al. 1999) (St. Louis, MO). All chemicals were of the highest grade were outlined based on morphological criteria (Duver- available. Drug solutions were made fresh daily before noy 1988). Brainstem and medullary regions were iden- each experiment. Drug stock solutions were prepared tified with the aid of an atlas (Paxinos and Huang 1995). (0.01 M) in either distilled water or 10 mM acetic acid Total binding was determined by incubating sections depending on their solubilities and were serially di- with 50 nM [3H]-clonidine without any competitors. In luted in the reaction buffer. ␣ adjacent sections, binding to 2AR was determined by subtracting the binding of [3H]-clonidine in the pres- Technical Note ence of 60 ␮M norepinephrine (NE) from the total bind- ing of [3H]-clonidine. Binding to I-sites was determined Several differences are noteworthy comparing our by subtracting the binding of [3H]-clonidine in the pres- method to the autoradiographic method of De Vos and ence of 60 ␮M NE plus 20 ␮M cirazoline from the bind- coworkers (1994): 1) The concentration of [3H]-clonidine in 3 ␮ ing of [ H]-clonidine in the presence of 60 M NE. NE our study (50 nM) was nearer its KD concentration for exhibited negligible binding to I-sites under the condi- I-sites (57 nM), rather than 8 nM used by De Vos and co- tions of this assay (Figure 5). On the other hand, cirazo- workers; 2) MIDX (12.5 ␮M, instead of 2 ␮M used by De line is known to displace [125I]p-iodoclonidine from hu- Vos and coworkers) and NE (60 ␮M) were compared as ϭ man I1 sites (Ki 99 nM) (Piletz et al. 1996). The displacing agents to demonstrate that the adrenergic bind- 3 ␣ ␣ percentage of total binding that was specific binding ing of [ H]-clonidine was of the 2AR type, not 1AR, as ␣ Ϯ Ϯ ␣ (including I-sites and 2AR) was 72 1 % (means well as to ensure complete masking of 2AR in the case of S.E.M. of all brain regions from all subjects). Of this spe- I-sites; 3) In our study, non-specific binding was assessed cific binding, 54 Ϯ 1% was binding to I-sites and 46 Ϯ with 20 ␮M cirazoline rather than with 10 ␮M cirazoline, ␣ ␮ 1% was binding to 2AR. because we found 20 M cirazoline resulted in more com- Competition curves were generated from slide wipes plete inhibition of [3H]-clonidine binding (Figure 5); 4) using ligand concentrations from 10Ϫ10 to 10Ϫ4 M (11 Binding reactions were conducted for 60 min, as com- points in triplicate for each of 3–4 subjects). The mea- pared to 20 min by De Vos and colleagues (1994). This was 700 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

based on earlier binding studies (Piletz et al. 1991); and 5) Despite the overall correlation between the densities 3 ␣ De Vos and colleagues used the antagonist, [ H]-MIDX, to of 2AR and I-sites (Figure 2), there were distinct sub- ␣ ␣ assess 2AR binding. By contrast, we used the agonist regional differences in the distributions of 2AR and 3 ␣ [ H]-clonidine to assess I-sites and 2AR binding. I-sites in the brain. Highest binding to I-sites was in the principle nucleus of the inferior olive (130 Ϯ 18 fmol/ mg protein) wherein the amount of binding was 10-fold RESULTS higher than that in the white matter of the prefrontal cortex (Table 1, Figure 2). Other areas of high density 3 ␣ 3 The specific binding of [ H]-clonidine to 2AR and I- for [ H]-clonidine binding to I-sites included the poly- sites was quantified by autoradiography in 27 human morphic layer of the hippocampus, dorsal and median brain regions, including specific areas of prefrontal cor- raphe, dentate gyrus, hypothalamus, locus coeruleus, tex, hippocampus, caudate, hypothalamus, pons, and central gray, and the hypoglossal nucleus (Table 1). the medulla oblongata (Table 1). Comparison of [3H]- Lowest [3H]-clonidine binding to I-sites was in the ␣ clonidine binding to 2AR and I-sites reveals overlap- white matter of the prefrontal cortex, the striosomes of ping, yet distinctly different distributions. For example, the caudate, and the pyramidal tract of the medulla. The at the level of the medulla, there was considerable simi- distribution of I-sites contrasted the distribution of [3H]- ␣ ␣ ␣ larity between the distribution of I-sites and 2AR (Fig- clonidine binding to 2AR. The highest binding to 2AR ure 1). In fact, there was a significant linear correlation occurred in the molecular layer of CA1 of the hippo- 3 ␣ between the amounts of [ H]-clonidine binding to 2AR campus and in the hypothalamus. Moderate amounts and to I-sites when considering all brain regions in a of binding were observed in the locus coeruleus. ϭ 2 ϭ ␣ scatter plot (p .0003, r 0.385; Figure 2). To further illustrate the distinction between 2AR

3 ␣ Table 1. Binding of [ H]-Clonidine to I-Sites and 2-Adrenoceptors in Human Brain ␣ Imidazoline Sites 2-Adrenoceptors Brain Region (fmol/mg protein) (fmol/mg protein)

Prefrontal Cortex Layer 1 36.3 Ϯ 9.6 47.9 Ϯ 12.7 Layers 2–3–4 41.1 Ϯ 9.5 41.7 Ϯ 12.6 Layers 5–6 34.9 Ϯ 7.8 42.1 Ϯ 6.7 White matter 13.8 Ϯ 6.5 7.9 Ϯ 2.3 Hippocampus CA4 76.8 Ϯ 8.5 67.3 Ϯ 7.3 CA3 55.5 Ϯ 3.1 43.4 Ϯ 5.0 CA2 49.2 Ϯ 5.4 21.5 Ϯ 8.8 CA1 stratum molecular 56.0 Ϯ 11.1 91.6 Ϯ 18.8 CA1 stratum radiatum 48.2 Ϯ 13.9 34.1 Ϯ 7.8 CA1 stratum lacunosum 45.4 Ϯ 15.0 48.8 Ϯ 15.6 CA1 stratum pyramidale 46.7 Ϯ 12.4 30.5 Ϯ 3.1 Polymorphic layer 97.0 Ϯ 3.6 65.5 Ϯ 16.5 Dentate gyrus Granular layer 86.2 Ϯ 4.5 52.4 Ϯ 13.4 Molecular layer 76.2 Ϯ 6.0 58.0 Ϯ 12.2 Head of Caudate Medial caudate 32.9 Ϯ 9.6 24.5 Ϯ 10.9 Lateral caudate 27.4 Ϯ 5.0 23.3 Ϯ 11.1 Striasomes 19.6 Ϯ 5.4 12.4 Ϯ 3.7 Hypothalamus 88.9 Ϯ 12.7 69.6 Ϯ 6.2 Pons Locus coeruleus 77.7 Ϯ 10.8 45.1 Ϯ 9.4 Dorsal raphe (caudal) 96.7 Ϯ 14.8 59.8 Ϯ 9.8 Medial raphe 90.3 Ϯ 10.8 38.5 Ϯ 15.5 Central gray 70.5 Ϯ 14.0 56.8 Ϯ 7.3 Medulla Inferior olive, principle nucleus 130.3 Ϯ 17.6 43.4 Ϯ 16.3 Inferior olive, dorsal nucleus 68.0 Ϯ 18.2 32.8 Ϯ 18.8 Hypoglossal nucleus 81.4 Ϯ 11.2 34.3 Ϯ 4.4 Rostral ventrolateral region 51.3 Ϯ 23.0 11.6 Ϯ 2.0 Ϯ Pyramidal tract 21.8 Ϯ 6.3 9.0 4.0

Each value is the mean Ϯ SEM data from five subjects. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 Clonidine Binding Sites in Brain 701

3 ␣ Figure 1. Autoradiograms of the binding of 50 nM [ H]-clonidine to I-sites (left) and to 2-adrenoceptors (right) in human medulla. Left panel: the specific binding of [3H]-clonidine to I-sites generated by digital subtraction of an autoradiogram of [3H]-clonidine binding in the presence of NE (60 ␮M) plus cirazoline (20 ␮M) from an autoradiogram of the binding of [3H]- 3 ␣ clonidine in the presence of NE alone. Right panel: the specific binding of [ H]-clonidine to 2-adrenoceptors generated by digital subtraction of the autoradiogram of the binding of [3H]-clonidine in the presence of NE (60 ␮M) from the autoradio- gram of the binding of [3H]-clonidine alone. For both panels, the two autoradiograms used to produce the image were gen- erated from adjacent tissue sections. The calibration bar illustrates the relationship between the color spectrum and radioligand binding density. The arrows indicate the rostral ventrolateral region, based on human brain atlas maps. and I-sites, quantitative transepts were drawn through ference between these sites was most obvious in the ␣ specific binding images of hippocampi using an image molecular layer of CA1. There, binding to 2AR clearly analysis system (Figure 3). Amounts of binding across a exceeded that of I-sites by 4:1, whereas in other subre- ␣ transept through a subregion may differ from the levels gions of the hippocampus the ratios of 2AR:I sites outlined in the entire subregion (i.e., data in Table 1). In were closer to 1:1. the outlining method (Table 1), uneven binding densi- To rule out the possibility that the agonist-induced ␣ ties within subregions are averaged by the computer state of 2AR might in some way account for brain I- program. In the transept method (Figure 3), pixel by sites (e.g., an artifact of using the agonists, [3H]-cloni- ␣ ␮ pixel densities are recorded across the subregions. dine and NE), we defined 2AR with either NE (60 M) ␣ ␮ Therefore, quantitative transepts should allow a closer or the 2AR antagonist, MIDX (12.5 M). MIDX is a se- ␣ examination of complex regions, like the hippocampus, lective antagonist for 2AR with very low affinity for у ␮ where uneven binding distributions exist. Histograms the human brain I-sites (IC50 at I-sites 60 M) (De Vos of the transept densities of [3H]-clonidine binding to et al. 1994; Flamez et al. 1997). We observed that specific ␣ ␣ I-sites and 2AR are shown in Figures 3 and 4. The dif- binding to 2AR was only slightly greater using MIDX, and the specific binding to I-sites was only slightly lower using MIDX compared to the results with NE. Furthermore, the relative distributions of binding to ␣ 2AR and I-sites were identical using either of these competitors (Figure 4, Panel C). The near identity of these results with either displacing agent makes it ␣ highly unlikely that an 2AR affinity state could ac- count for I-sites. Competition curves were next generated for [3H]- clonidine binding (under a mask of 12.5 ␮M MIDX) at caudate I-sites (Figure 5). [3H]-Clonidine was found to ϭ Ϯ have a KD 57 3 nM for I-sites, in keeping with the results of De Vos and colleagues (1994). The head of the caudate was chosen for these studies because of the availability of tissue. Five compounds were capable of completely displacing [3H]-clonidine from caudate I-sites, but these mostly had shallow competition curves (Figure 5). The only exception was the ␤-carbo- Figure 2. Correlation between the binding of [3H]-cloni- ␣ line, harmane, which exhibited a monophasic competi- dine to I-sites and to 2-adrenoceptors in 27 brain regions from human prefrontal cortex, hippocampus, caudate, tion curve (Figure 5). The rank order of IC50 values for hypothalamus, pontine region, and the medulla oblongata I-sites (generated using a single site model) was as fol- (p ϭ .0003, r2 ϭ 0.385). Each point represents the mean value lows: cirazoline Ͼ harmane Ͼ BDF6143 Ͼ idazoxan ϭ of binding obtained for each region (five subjects/region). tizanidine. Agmatine, efaroxan, moxonidine, NE, and 702 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

3 ␣ Figure 3. Comparison of the distribution of the binding of [ H]-clonidine to I-sites (A) and to 2-adrenoceptors (B) in the ␣ 3 human hippocampus using norepinephrine (NE) to identify 2-adrenoceptors. (A): The specific binding of [ H]-clonidine to I-sites was generated by digital subtraction of an autoradiogram of [3H]-clonidine binding in the presence of NE (60 ␮M) plus cirazoline (20 ␮M) from an autoradiogram of the binding of [3H]-clonidine in the presence of NE alone. (B): The specific 3 ␣ 3 binding of [ H]-clonidine to 2-adrenoceptors generated by digital subtraction of the autoradiogram of the binding of [ H]- clonidine in the presence of NE (60 ␮M) from the autoradiogram of the binding of [3H]-clonidine alone. For both images, the two autoradiograms used to produce the image shown were generated from adjacent tissue sections. White outline bars overlaid upon the images of A and B indicate where a quantitative transept bar was placed. (C): The quantitative distribu- 3 ␣ tions of [ H]-clonidine binding to I-sites (lower line) and to 2-adrenoceptors (upper line in 4), expressed pixel by pixel along the horizontal axis. Representative regions along these transepts are indicated by numbers: 1 ϭ CA3; 2 ϭ CA4; 3 ϭ polymor- phic area; 4 ϭ Molecular layer of CA1; 5 ϭ transitional area between CA1 and subiculum.

3 oxymetazoline did not inhibit [ H]-clonidine binding rank-order for human I2 sites in homogenate binding Ͼ ϾϾ Ͼ sufficiently to determine IC50 values. Actually, agma- assays is cirazoline idazoxan oxymetzoline tine failed to displace any I-sites labeled by [3H]-cloni- moxonidine у efaroxan Ͼ clonidine (Piletz and Sletten dine in caudate (Figure 5, Table 2). 1993). Thus, the I-site(s) under study has(have) a For comparative purposes, Table 2 shows our Ki val- unique pharmacological profile. ues beside those previously determined by De Vos and Moxonidine, a compound with preferential I1 affinity, colleagues (1994), who also used the head of the human was found to have low affinity in caudate sections for caudate for competition binding studies. The rank-order I-sites labeled by [3H]-clonidine (Figure 5, Table 2). To ex- for human I1 sites in homogenate binding assays is amine this further, moxonidine was used in parallel with known to be moxonidine у oxymetazoline Ͼ tizanidine cirazoline to define non-adrenergic binding of [3H]-cloni- у agmatine у efaroxan ϭ clonidine Ͼ cirazoline Ͼ dine in a number of regions of the medulla from three BDF6143 ϾϾ idazoxan ϾϾ NE (Piletz et al. 1996). The subjects. The RVLM is often cited as mediating the non- NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 Clonidine Binding Sites in Brain 703

3 ␣ Figure 4. Comparison of the distribution of the binding of [ H]-clonidine to I-sites (A) and to 2-adrenoceptors (B) in the ␣ 3 human hippocampus using methoxy-iodazoxan (MIDX) to identify 2-adrenoceptors. (A): The specific binding of [ H]-cloni- dine to I-sites was generated by digital subtraction of an autoradiogram of [3H]-clonidine binding in the presence of MIDX (12.5 ␮M) and cirazoline (20 ␮M) from an autoradiogram of the binding of [3H]-clonidine in the presence of MIDX alone. (B): 3 ␣ The specific binding of [ H]-clonidine to 2-adrenoceptors generated by digital subtraction of the autoradiogram of the bind- ing of [3H]-clonidine in the presence of MIDX (12.5 ␮M) from the autoradiogram of the binding of [3H]-clonidine alone. For both images, the two autoradiograms used to produce the image shown were generated from adjacent tissue sections. White outline bars overlaid upon the images of A and B indicate where a quantitative transept bar was placed. (C): The quantita- 3 ␣ tive distributions of [ H]-clonidine binding to I-sites (lower line) and to 2-adrenoceptors (upper line), expressed pixel by pixel along the horizontal axis. Representative regions along these transepts are indicated by numbers: 1 ϭ CA3; 2 ϭ CA4; 3 ϭ polymorphic layer; 4 ϭ Molecular layer of CA1; 5 ϭ transitional area between CA1 and subiculum. adrenergic hypotensive effects of clonidine, moxonidine, ence of NE plus moxonidine, but was significantly greater and rilmenidine (Chan et al. 1996). Table 3 shows four than the amount of binding remaining in the presence of conditions of [3H]-clonidine binding: (a) without any NE plus cirazoline (Table 3). Even in the RVLM (Figure 1, competitors (total binding); (b) in the presence of 60 ␮M arrow), [3H]-clonidine binding was not substantially more ␣ ␮ NE (displacing 2AR); (c) in the presence of 60 M NE displaced by moxonidine than NE (Table 3). ␮ ␣ plus 20 M cirazoline (displacing 2AR and I-sites); and (d) in the presence of 60 ␮M NE plus 5 ␮M moxonidine ␣ (displacing 2AR and only a small additional amount of DISCUSSION I-sites). The amount of [3H]-clonidine binding in any re- gion remaining in the presence of NE was not signifi- In the present study, the distributions of [3H]-clonidine ␣ cantly different from the amount remaining in the pres- binding to I-sites and 2AR were distinct (Figures 1, 3, 704 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

Figure 5. Inhibition of the binding of [3H]-clonidine (50 nM) to I-sites in consecutive human caudate section wipes by imi- 3 ␣ dazoline and non-imidazoline compounds. In all cases, the binding of [ H]-clonidine to 2-adrenoceptors was prevented by adding 12.5 ␮M methoxy-idazoxan to the binding reaction mixture. Competing ligands were (᭢) agmatine, (᭛) NE, (᭿) moxonidine, (᭺) oxymetazoline, (᭡) BDF6143, (᭞) idazoxan, (᭜) tizanidine, (ᮀ) harmane, and (᭹) cirazoline. Each point represents the mean Ϯ S.E.M. of values generated from three separate experiments. and 4) but correlated across 27 brain regions (Figure 2). that the non-adrenergic site labeled by [3H]-clonidine in

As expected, the RVLM (Figure 1, arrow) had a high ra- the human RVLM is of the I1 subtype (see below). ␣ tio of I-sites: 2AR (nearly 5:1) compared to other brain A clear distinction in the regional distribution of 3 ␣ regions (Table 1). This is noteworthy because the RVLM [ H]-clonidine labeling to 2AR and I-sites was obvious is the site of the anti-hypertensive action ascribed to IR1 in the hippocampus (see Figures 3 and 4). The distribu- ␣ stimulation when imidazolines are micro-injected into tion of I-sites and 2AR was virtually identical whether the brainstem (Bousquet et al. 1984). However, a number defined by masking [3H]-clonidine binding with an ␣ ␣ of brain regions were more abundantly enriched with 2AR agonist (NE) or an 2AR antagonist (MIDX; Fig- I-sites than the RVLM, notably the inferior olive, pontine ures 3 and 4). Approximately 50% of the [3H]-clonidine regions, and hypothalamus (Table 1). Furthermore, our binding in the human hippocampal CA1 molecular ␣ ഠ competition binding curves (Figure 5) make it doubtful layer was to 2AR. This compared to 35% binding to I-sites and ഠ15% non-specific binding in the same CA1 region. Table 2. Ki Values for I-Sites in Caudate Section Wipes It should be noted that ␣ AR would be nearly satu- Labeled with 50 nM [3H]-Clonidine. 2 rated at a concentration of 50 nM [3H]-clonidine, since ␣ Ligand DeVos et al., (1994) Present Findings clonidine has a high affinity at each of the human 2AR ␣ ϭ ␣ ϭ ␣ ϭ ( 2AAR, KD 1.5 nM; 2BAR, KD 7.8 nM; 2CAR, KD Cirazoline 8.4 Ϯ 0.5 nM 88.3 Ϯ 2.1 nM 125 Ϯ 1.3 nM for [ I]p-iodoclonidine) (Piletz et al. 1996). On Guanoxan 56 8— ϭ Rilmenidine 214 Ϯ 36 — the other hand, I-sites would only be half-saturated (KD Harmane — 521 Ϯ 12 55 nM for [125I]p-iodoclonidine). Therefore, the propor- BDF6143 — 693 Ϯ 19 tions of these two sites in, for instance, the CA1 molecu- Idazoxan 20.7 Ϯ 1.1 1330 Ϯ 32 lar layer might actually be re-estimated to be 6:5 for Ϯ UK14,304 770 10 — I-sites:␣ AR. Using similar calculations, the CA4 region ST587 950 Ϯ 13 — 2 Tizanidine — 1540 Ϯ 62 of the hippocampus might be estimated to possess a 9:3 a ␣ 3 Oxymetazoline — Ͼ36,200 ratio of I-sites: 2AR as labeled by [ H]-clonidine. Thus, Ͼ a ␣ Norepinephrine — 50,400 I-sites and 2AR appeared unevenly distributed in the Moxonidine — Ͼ55,600a hippocampus with each site having a unique distribution. Ͼ a Efaroxan — 74,700 A variety of previous autoradiographic studies of Agmatine — Ͼ100,000 I-sites, mostly in animals, exist in the literature (De Vos a In these cases the non-linear regression fits were forced through y ϭ 0. et al. 1994; Ernsberger et al. 1994; King et al. 1995; Lione NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 Clonidine Binding Sites in Brain 705

Table 3. Binding of [3H]-Clonidine in Subregions of the Human Medulla in the Absence and Presence of Non-Imidazoline and Imidazoline Compoundsa

[3H]-Clonidine Binding in the Presence of: Total [3H]-clonidine Norepinephrine Norepinephrine Brain Regionbinding Norepinephrine and Cirazoline and Moxonidine

Inferior olive, principle nucleus 270Ϯ12 226Ϯ20 96Ϯ19 199Ϯ17 Inferior olive, dorsal nucleus 180Ϯ23 148Ϯ480Ϯ21 140Ϯ15 Hypoglossal nucleus 150Ϯ11 116Ϯ935Ϯ4 110Ϯ1 Rostral ventrolateral region 129Ϯ5 117Ϯ789Ϯ41 103Ϯ6 Pyramidal tract 50Ϯ841Ϯ10 20Ϯ644Ϯ9

aConcentrations of inhibitors were: norepinephrine, 60 ␮M; cirazoline, 20 ␮M; moxonidine, 5 ␮M. Each value (in fmol/mg protein) is the mean Ϯ SEM of determinations made in three subjects. et al. 1998; Mallard et al. 1992). Unfortunately, the re- NE or MIDX were used to displace [3H]-clonidine from sults of those previous studies have been as diverse as adrenergic sites, and cirazoline was used (under NE or the radioligands used to define them. Besides us, only MIDX masks) to displace [3H]-clonidine from non- De Vos and colleagues (1994) have used [3H]-clonidine adrenergic I-sites. Nearly identical images were ob- to visualize brain I-sites. Our results compare favorably tained with either NE or MIDX as displacers (Figures 3 in a number of ways with theirs (Table 2). On the other and 4). Therefore, any hypothesis invoking an agonist- 3 ␣ hand, Ernsberger and coworkers (1994) reported dif- induced state of [ H]-clonidine binding to 2AR in or- 125 fuse [ I]p-iodoclonidine binding to “I1-like” sites in rat der to explain the existence of I-sites seems unlikely. ␣ brainstem sections. Their diffuse images contrasted Equally important, cirazoline is an imidazoline 1AR ␣ sharply with the highly regionalized images of Figures agonist without high affinity for known human 2AR ␣ 1, 3, and 4. For instance, the striking intensity of I-sites subtypes (Ki values for 2AR subtypes vary from 193 to in the olivary nuclei (Figure 1) appears to be a “signa- 657 nM) (Piletz et al. 1996). At 50 nM, [3H]-clonidine 3 ␣ ture” for human I-sites labeled with [ H]-clonidine, but would bind minimally to 1AR because the Ki value of 125 ␣ this was not reported for [ I]p-iodoclonidine autorad- clonidine for brain 1AR is 750 nM (Glossmann et al. iograms (Ernsberger et al. 1994). We believe this dis- 1981). crepancy from the results with [125I]p-iodoclonidine re- [3H]-cirazoline has previously been shown to label a ported by Ernsberger and coworkers (1994) may be non-adrenergic/ non-I1 / non-I2 site in brain homoge- related to the lipophilicity of [125I]p-iodoclonidine (see nate binding assays (Angel et al. 1995). In characteriz- below). There are also autoradiographic images from ing that I-site, Angel et al. (1995) reported the Ki values rat brain using [3H]idazoxan (Mallard et al. 1992), of several of the same drugs studied here. They re- [3H]rilmenidine (King et al. 1995), and [3H]2-(2-benzo- ported a rank ordering of affinities for I-sites of cirazo- furanyl-2-imidazoline (Lione et al. 1998). The latter line (Ki ϭ 4 nM) Ͼ clonidine (Ki ϭ 50 nM) Ͼ idazoxan ϭ ϾϾ Ͼ three studies have all related their findings to I2 sites (Ki 158 nM) oxymetazoline (22,000 nM) efar- (King et al. 1995; Lione et al. 1998; Mallard et al. 1992). oxan (219,000 nM) Ͼ (-) epinephrine (635,000 nM). This By convention, I-sites labeled with [3H]-clonidine rank-order of Ki values corresponds closer to our data have been designated as I1 sites (Ernsberger et al. 1987). (Table 2) than any other I-sites reported. Therefore, the However, De Vos and colleagues (1994) used caution- same receptor(s) may have been under study by [3H]- ary language when making an assignment of their non- clonidine displacement with cirazoline (in our study) 3 3 adrenergic [ H]-clonidine binding site as an I1 subtype. and by [ H]-cirazoline displacement with clonidine They reported that idazoxan and guanoxan had higher (Angel et al. 1995). The identity and function of the 3 affinities than expected for an I1 site (Table 2), but cloni- [ H]-cirazoline-sensitive I-site studied by Angel and co- dine and rilmenidine had affinities consistent with an I1 workers (1995) remains unknown. subtype. In that same study, they clearly distinguished De Vos and colleagues (1994) found no correlation 3 3 ␣ their site from the I2 site labeled by [ H]-idazoxan and between I-sites labeled with [ H]-clonidine and 2AR ␣ 3 3 2 ϭ Ϫ from the 2AR labeled by [ H]-MIDX (De Vos et al. sites labeled with [ H]-MIDX (r 0.068) when com- 1994). Therefore, De Vos and colleagues concluded: “At paring the densities of these two sites in human brain first glance, IR appear to correspond to the I1 imidazo- regions. This is a surprising difference from the positive line receptor subtype . . . however, this comparison is correlation we observed (p ϭ .0003, r2 ϭ 0.385) across 27 subject to some caution.” Their cautionary note is ech- brain regions between these sites (Figure 2). However, oed by our present results. of the eight brain regions in common between our two Special emphasis may be given to our choice of cira- studies (cortical layers 1, 2–4, 5–6, hippocampal layer zoline for defining specific binding to I-sites (Figure 5). CA1, dentate gyrus, caudate putamen, raphe nuclei, 706 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

and olivary nucleus), we also found no correlation be- ent from those recommended for filtration binding as- 3 ␣ tween I-sites labeled with [ H]-clonidine and 2AR sites says of homogenate preparations of I1 sites (Ernsberger labeled with [3H]-clonidine (p ϭ .58, r2 ϭ 0.053). There- et al. 1995b). Optimal buffer conditions for homogenate fore, the discrepancy between our studies could simply I1 sites invoke low osmotic and ionic strengths (Erns- be due to different brain regions under study. It should berger et al. 1995b). Furthermore, purified plasma be emphasized that De Vos and coworkers (1994) used membranes are considered useful to lower the signal- ␣ 3 the selective 2AR-antagonist, [ H]-MIDX, to measure to-noise ratio of I1 sites in most filtration binding assays ␣ ␣ 2AR binding, while we used the mixed 2AR/I-site ag- of I1 sites (Ernsberger et al. 1995b). Also, a high flow- onist [3H]-clonidine. through vacuum system is considered critical for filtra- ␣ De Vos and coworkers (1994) did not report on 2AR tion binding assays of I1 sites, due to their fast off-rate binding with [3H]-clonidine, although they used it for I- of binding (Piletz et al. 1991). Hypo-osmolarity and hy- sites. Agonists, like [3H]-clonidine, preferentially in- potonic buffer conditions could not be used autoradio- ␣ duce a high affinity state of the 2AR (Alburges et al. graphically, because these conditions destroy the ana- 1993), whereas antagonists bind equally to low and tomical integrity of slide-mounted tissue sections. ␣ high affinity states of 2AR. This difference in the states Hence, comparisons of affinities derived from autorad- ␣ 3 of 2AR labeled by [ H]-clonidine (high affinity states), iographic binding studies and homogenate binding versus both low and high affinity states in their study studies may not be legitimate for I1 or I2 sites. It remains (De Vos et al. 1994) might underlie the different correla- a possibility that the different conditions used in the tions between our studies. For instance, the correlation present study might have significantly altered the affin- observed in Figure 2 may in fact relate to G-proteins in ities of certain ligands, such as moxonidine, for true ␣ common with 2AR and I-sites. I1-sites. The subtype or subtypes of I-sites labeled by [3H]- Mention should also be made that most previous ␣ clonidine in human brain remains open to question. In membrane binding studies of I1 and 2AR sites (Piletz competition studies with human caudate sections, we and Sletten 1993; Klimek et al. 1999) have used [125I]-p- observed the affinities of I-sites labeled by [3H]-cloni- iodoclonidine as radioligand. In preliminary studies, 125 dine were not consistent with either an I1 or I2 site (Ta- we found that if [ I]-p-iodoclonidine was substituted 3 ble 1). Three potent I1 ligands (efaroxan, oxymetazoline, for [ H]-clonidine using the autoradiographic method, and moxonidine) did not displace [3H]-clonidine bind- a substantial amount of [125I]-p-iodoclonidine binding ing from caudate I-sites, even at relatively high concen- was localized to white matter (data not shown). Al- trations (Table 1). The possibility that the I-sites labeled though [3H]-para-aminoclonidine and [125I]-p-iodoclo- 3 by [ H]-clonidine are actually I2 sites also is unlikely be- nidine are known to have similar properties (Alburges cause higher than expected affinities were observed for et al. 1993), we found little or no specific labeling of several agents (i.e., BDF6143 and rilmenidine affinities white matter with [3H]-clonidine. Therefore, even were 4-fold and 54-fold higher, respectively, than ex- closely related imidazoline radioligands, like [3H]- 125 pected for human I2 sites) (Table 2) (Diamant et al. clonidine and [ I]-p-iodoclonidine, might bind differ- 1992). Furthermore, [3H]-clonidine binding was exam- ent sites. ined in human medullary slices with the goal of identi- The molecular basis of imidazoline receptors is an- fying a typical I1 site (i.e., moxonidine-displaceable). A other area of intense research. There is little doubt that series of medullary slices that included the human the diversity of ligand-defined subtypes will be RVLM were studied (Figure 1, arrow). Our autoradio- matched by protein subtypes. A cDNA clone which en- graphic technique was the same as in caudate, except, codes a peptide with I1-like properties has recently been in addition to the presence of 20 ␮M cirazoline in some described (Ivanov et al. 1998; Piletz et al. 2000a) and, slices, 5 ␮M moxonidine was added to measure the surprisingly, the predicted protein has a novel amino non-specific binding in adjacent sections. Yet, moxoni- acid sequence, different from known receptors. There- dine competed for only slightly more total sites than fore, it is uncertain if the cloned I1 site is actually a true NE in the medullary slices (Table 3). Oxymetazoline receptor (Piletz et al. 2000a). I2 sites are somewhat better was also ineffective at displacing [3H]-clonidine from understood, and have been physically linked to I-sites in the medulla (data not shown). Moxonidine monoamine oxidases (Tesson et al. 1995). In addition, was also found to be an ineffective displacer of I-sites in there are at least two subtypes of I2 sites that exhibit bi- the hippocampus and prefrontal cortex (data not phasic binding curves (Eglen et al. 1998). An I3 site has shown). Thus, we found no autoradiographic evidence been hypothesized to reside on the ion conducting pore 3 for typical I1 sites labeled by [ H]-clonidine, even in the component of the ATP-sensitive potassium channel medulla. (Monks et al. 1999). To these sites we now add another It should also be mentioned that the conditions in I-site labeled similarly by [3H]-cirazoline (Angel et al. our autoradiographic procedure, while routine for 1995) and [3H]-clonidine (present study). many receptors (Klimek et al. 1997), were vastly differ- Harmane had high affinity (Ki ϭ 521 nM) for I-sites NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6 Clonidine Binding Sites in Brain 707

3 ␣ labeled by [ H]-clonidine in the human caudate (Figure (1995): Role of medullary I1-imidazoline and 2-adren- 2). This high affinity Ki value supports the candidacy of ergic receptors in the antihypertensive responses harmane as an endogenous I-site ligand (Hudson 1999). evoked by central administration of clonidine analogs in conscious spontaneously hypertensive rats. J Phar- Harmane has been reported to have high affinity for I1 macol Exp Ther 273:1162–1171 sites in filtration binding assays and to be active in Chan CK, Sannajust F, Head GA (1996): Role of imidazoline modulating blood pressure in the CNS (Hudson 1999). receptors in the cardiovascular actions of moxonidine, The other endogenous I-site ligand, agmatine, had no rilmenidine and clonidine in conscious rabbits. J Phar- affinity whatsoever for I-sites in the present study. Lit- macol Exp Ther 276:411–420 tle is known about the regional distribution of harmane De Vos H, Bricca G, DeKeyser J, DeBacker J, Bousquet P, Vau- in the brain, and future studies will be required to deter- quelin G (1994): Imidazoline receptors, non-adrenergic ␣ mine if it co-localizes with putative IR sites. idazoxan binding sites, and 2 adrenoceptors in the In summary, the distribution of a novel I-site for human central nervous system. Neuroscience 59:589–598 [3H]-clonidine has been described in human brain. The Diamant S, Eldar-Geva T, Atlas D (1992): Imidazoline bind- pharmacology of this site is in agreement with a [3H]- ing sites in human placenta: Evidence for heterogeneity clonidine binding site described by De Vos and co- and a search for physiological function. Br J Pharmacol 106:101–108 workers (1994) and a [3H]-cirazoline site described by Angel and coworkers (1995). It remains to be discov- Duvernoy H (1988): The Human Hippocampus. An Atlas of ered what the function of this site is, but given the Applied Anatomy. JF Bergmann, Munich. plethora of actions previously ascribed to clonidine Eglen RM, Hudson AL, Kendall DA, Nutt DJ, Morgan NG, (Buccafusco 1992), it should be of interest to psychiatry. Wilson VG, Dillon MP (1998): ‘Seeing through a glass darkly’: Casting light on imidazoline ‘I’ sites. Trends Currently, we are attempting to determine whether this Pharmacol Sci 19:381–390 is the same I-site that was reported previously to be de- Ernsberger P, Graves ME, Graff LM, Zakieh N, Nguyen P, creased in the hippocampus of depressed suicide vic- Collins LA, Westbrooks KL, Johnson GG (1995a): I1-Imi- tims (Piletz et al. 2000b) and increased in the frontal cor- dazoline receptors: Definition, characterization, distri- tex of depressed suicide victims (Garcia-Sevilla et al. bution and transmembrane signalling. Ann NY Acad 1996). Sci 763:22–42

Ernsberger P, Haxhiu MA (1997): The I1-imidazoline-bind- ing site is a functional receptor mediating vasodepres- ACKNOWLEDGMENTS sion via the ventral medulla. Am J Physiol 42:R1572– R1579 We are grateful to Josh Farley (University of Mississippi Med- Ernsberger P, Haxhiu MA, Graff LM, Collins LA, Dreshaj I, ical Center) and Jane Schenck (University of Mississippi Med- Grove DL, Graves ME, Schafer SG, Christen MO (1994): ical Center) for their technical assistance, and to Dr. Violetta A novel mechanism of action for hypertension control: Klimek for her valuable suggestions. The authors gratefully Moxonidine as a selective I1 imidazoline agonist. Car- acknowledge the assistance of Dr. Craig Stockmeier (Univer- diovasc Drugs Ther 8:27–41 sity of Mississippi Medical Center), Lisa Konick (Case West- Ernsberger P, Meeley MP, Mann JJ, Reis DJ (1987): Clonidine ern Reserve University), and Dr. Balraj and staff at the Cuya- ␣ binds to imidazole binding sites as well as 2-adreno- hoga County Coroner’s office, Cleveland, Ohio. This research ceptors in the ventrolateral medulla. Eur J Pharmacol was supported by NIH grants MH42859, MH46692, and 134:1–13 MH45488. Ernsberger P, Piletz JE, Graff LM, Graves ME (1995b): Opti- mization of radioligand binding assays for I1 imidazo- line sites. Ann NY Acad Sci 763:163–168 REFERENCES Flamez A, De Backer J-P, Czerwiec E, Ladure P, Vauquelin G (1997): Pharmacological characterization of I and I ␣ 1 2 Alburges ME, Bylund DB, Pundt LL, Wamsley JK (1993): 2 imidazoline receptors in human striatum. Neurochem agonist binding sites in brain: [125I]para-iodoclonidine Int 30:25–30 versus [3H]para-aminoclonidine. Brain Res Bull 32:97–102 Garcia-Sevilla JA, Escriba PV, Sastre M, Walzer C, Busquets Angel I, Le Rouzic M, Pimoule C, Graham D, Arbilla S X, Jaquet G, Reis DJ, Guimon J (1996): Immunodetection 3 (1995): [ H]cirazoline as a tool for the characterization of and quantitation of imidazoline receptor proteins in imidazoline sites. Ann N Y Acad Sci 763:112–124 platelets of patients with major depression and in brains Bousquet P, Feldman J, Schwartz J (1984): Central cardiovas- of suicide victims. Arch Gen Psychiatry 53:803–810 cular effects of alpha adrenergic drugs: Differences Glossmann H, Lubbecke F, Bellemann P (1981): [125I]-HEAT, between catecholamines and imidazolines. J Pharmacol a selective, high-affinity, high specific activity ligand for Exp Ther 230:232–236 ␣ 1 adrenoceptors. Naunyn Schm Arch Pharmacol 318:1–9 Buccafusco JJ (1992): Neuropharmacologic and behavioral Hudson A, Price R, Tyacke RJ, Lalies MD, Parker CA, Nutt actions of clonidine: Interactions with central neu- DJ (1999): Harmane, norharmane and tetrahydro beta- rotransmitters. Int Rev Neurobiol 33:55–107 carboline have high affinity for rat imidazoline binding Buccafusco JJ, Lapp CA, Westbrooks KL, Ernsberger P sites. Br J Pharmacol 126:2P 708 J.E. Piletz et al. NEUROPSYCHOPHARMACOLOGY 2000–VOL. 23, NO. 6

Ivanov TR, Jones JC, Dontenwill M, Bousquet P, Piletz JE Piletz JE, Andorn AC, Unnerstall JR, Halaris A (1991): Bind- 3 ␣ (1998): Characterization of a partial cDNA clone ing of [ H]-p-aminoclonidine to 2-adrenoceptor states detected by imidazoline receptor-selective antisera. J plus a non-adrenergic site on human platelet plasma Auton Nerv Syst 72:98–110 membranes. Biochem Pharmacol 42:569–584 King PR, Gundlach AL, Louis WJ (1995): Quantitative auto- Piletz JE, Ivanov TR, Sharp JD, Ernsberger P, Chang C-H, ␣ radiographic localization in rat brain of 2-adrenergic Pickard RT, Gold G, Roth B, Zhu H, Jones JC, Baldwin J, and non-adrenergic I-receptor binding sites labeled by Reis DJ (2000a): Imidazoline receptor antisera-selected [3H]rilmenidine. Brain Res 675:264–278 (IRAS) cDNA: Cloning and characterization. DNA Cell Biol 19:319–330 Klimek V, Rajkowska G, Luker SN, Dilley G, Meltzer HY, Overholser JC, Stockmeier CA, Ordway GA (1999): Piletz JE, Sletten K (1993): Nonadrenergic imidazoline bind- Brain noradrenergic receptors in major depression and ing sites on human platelets. J Pharmacol Exp Ther schizophrenia. Neuropsychopharmacology 21:69–81 267:1493–1502 Klimek V, Stockmeier C, Overholser J, Meltzer HY, Kalka S, Piletz JE, Zhu H, Chikkala DN (1996): Comparison of ligand binding affinities at human I1 imidazoline binding sites Dilley G, Ordway GA (1997): Reduced levels of norepi- ␣ nephrine transporters in the locus coeruleus in major and the high affinity state of 2 adrenoceptor subtypes. J depression. J Neurosci 17:8451–8458 Pharmacol Exp Ther 279:694–702 Lione LA, Nutt DJ, Hudson AL (1998): Characterisation and Piletz JE, Zhu H, Ordway G, Stockmeier C, Dilly G, Reis D, localization of [3H]2-(2-benzofuranyl)-2-imidazoline Halaris A (2000b): Imidazoline receptor proteins in the binding in rat brain: A selective ligand for imidazoline hippocampus of major depressives. Biol Psychiat 48(9) I2 receptors. Eur J Pharmacol 353:123–135 Raddatz R, Lanier SM (1997): Relationship between imida- zoline/guanidinium receptive sites and monoamine Mallard NJ, Hudson AL, Nutt DJ (1992): Characterization oxidase A and B. Neurochem Int 30:109–117 and autoradiographical localization of non-adrenocep- tor idazoxan binding sites in the rat brain. Br J Pharma- Regunathan S, Feinstein DL, Reis DJ (1999): Anti-prolifera- col 106:1019–1027 tive and anti-inflammatory actions of imidazoline agents. Are imidazoline receptors involved? Ann N Y McPherson CA (1985): Analysis of radioligand binding Acad Sci 881:410–419 experiments. A collection of computer programs for the IBM PC. J Pharmacol Methods 14:213–228 Reis DJ, Regunathan S (1999): Agmatine: An endogenous ligand at imidazoline receptors is a novel neurotrans- Michel MC, Ernsberger P (1992): Keeping an eye on the I mitter. Ann N Y Acad Sci 881:65–80 site: Imidazoline-preferring receptors. Trends Pharma- col Sci 13:369–370 Tesson F, Limon-Boulez I, Urban P, Puype M, Vandekerck- hove J, Coupry I, Pompon D, Parini A (1995): Localization Molderings GJ (1997): Imidazoline receptors: Basic knowl- of I2-imidazoline binding sites on monoamine oxidases. edge, recent advances and future prospects for therapy. J Biol Chem 270:9856–9861 Drugs Future 22(7):757–772 van Zwieten PA (1997): Central imidazoline (I1) receptors as Monks LK, Cosgrove KE, Dunne MJ, Ramsden CA, Morgan targets of centrally acting antihypertensives: Moxoni- NG, Chan SLF (1999): Affinity isolation of imidazoline dine and rilmenidine. J Hypertens 15:117–125 binding proteins from rat brain using 5-amino-efaroxan Vandergriff J, Kallman MJ, Rasmussen K (2000): Moxonidine, as a ligand. FEBS Lett 447:61–64 a selective imidazoline-1 receptor agonist, suppresses Paxinos G, Huang X-F (1995): Atlas of the Human Brain- the effects of ethanol withdrawal on the acoustic stem. Academic Press, San Diego, CA startle response in rats. Biol Psychiat 47:874–879