Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and ␣ 2-Adrenergic Receptors in Human Brain John E

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.

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