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Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs
Brain Research 770 Ž1997. 261–266
Research report
Autoradiographic studies of central a 2A- and a 2C-adrenoceptors in the rat using w 3 HxMK912 and subtype-selective drugs Staffan Uhlen ´ a
a, )
, Jonas Lindblom a , Allan Johnson b , Jarl E.S. Wikberg
a
Department of Pharmaceutical Biosciences, DiÕision of Pharmacology, Box 591, Uppsala UniÕersity, S-751 24 Uppsala, Sweden b Department of Psychiatry, Uppsala UniÕersity, Uppsala, Sweden Accepted 1 July 1997
Abstract In the present study we examined the distribution of a 2A- and a 2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between a 2A- and a 2C-adrenoceptors, the slices were incubated with w 3 HxMK912 in the presence of graded concentrations of the a 2A-selective drug, BRL44408, or the a 2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM w 3 HxMK912 plus 185 nM BRL44408 selectively labeled a 2C-adrenoceptors, while 0.4 nM w 3 HxMK912 plus 220 nM spiroxatrine selectively labeled a 2A-adrenoceptors. Using this approach, a 2C-adrenoceptors were detected in the striatum, while a 2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus. q 1997 Elsevier Science B.V. Keywords: Autoradiography; a 2A -Adrenoceptor; a 2C -Adrenoceptor; w 3 HxMK912; Spinal cord; Striatum
1. Introduction Based on structural and pharmacological evidence, the a 2-adrenoceptors are currently classified into three subtypes designated a 2A -, a 2B - and a 2C -adrenoceptors w3x. The rat and the human orthologs of the a 2A -adrenoceptor show somewhat discrepant pharmacological properties, and sometimes the rat ortholog is designated a 2D . Still the consensus of today is that there exist only three a 2-adrenoceptor subtypes within one species w9x. In radioligand binding studies performed on membranous homogenates, and using the 20-fold a 2C - to a 2A selective radioligand w 3 HxMK912, both a 2A - and a 2C adrenoceptors were detected in the rat cerebral cortex w25x, striatum, and at different levels of the spinal cord w27x. Similarly, a 2A - and a 2C -adrenoceptors were detected in membranes from the human caudate nucleus using either w 3 Hxyohimbine w17x or w 3 HxRX821002 w21x. A more precise anatomical localization of a 2-adrenoceptors has been performed in nervous tissue using autoradiography. Thus, a 2-adrenoceptors were visualized in different sections of the rat brain and spinal cord using
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Corresponding author. Fax: q46 Ž18 . 559-718; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 6 0 - 3
w 3 H x p-aminoclonidine w28x, w 3 H xrauwolscine w2,20x, w 3 Hxidazoxan w2x and w 3 Hxrilmenidine w10x. Autoradiographic studies with w 3 Hxrauwolscine, w 3 Hxyohimbine and w 3 HxRX821002 have given further information on the distribution of a 2-adrenoceptors in the human brain and spinal cord w4,8,17x. However, in none of these autoradiographic studies any attempt was made to characterize which a 2-adrenoceptor subtypes became labeled. Northern blot analysis indicates that mRNAs encoding the a 2A - and a 2C -adrenoceptors were widely distributed in the rat CNS whereas a 2B -encoding mRNA was detectable only in the diencephalon w30x. Moreover, using in situ hybridization it was shown that mRNAs encoding both a 2A - and a 2C -adrenoceptors are present in the cerebral cortex, cerebellum, spinal cord, and other parts of the rat nervous system w14–16x. Drugs active on a 2-adrenoceptors are in clinical use, or may find clinical use in the near future, as antidepressants, for the induction of anesthesia or analgesia, for the treatment of glaucoma Žsee w7,11x., for the treatment of attention-deficit hyperactivity disorders w1x, for treatment of hypertension w13x, and in the field of Parkinson’s disease w6,23x. Characterization of the specific a 2-adrenoceptor subtypes involved in the different physiological effects mediated through the a 2-adrenoceptor seems warranted. The present study was undertaken to develop an ap-
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proach for the delineation of a 2A - and a 2C -adrenoceptors in the rat central nervous system using receptor autoradiographic techniques. Our results with w 3 HxMK912 in conjunction with subtype-selective drugs show that a 2C adrenoceptors are detectable in the striatum, while a 2A adrenoceptors are detectable in the somatosensory frontoparietal cerebral cortex, the endopiriform nucleus, the septum and the spinal cord distal dorsal horn and ventral horn. 2. Materials and methods
2.2. Radioligands, drugs and chemicals w 3 HxMK912 ŽŽ2 S,12bS .-1X ,3X-dimethylspiroŽ1,3,4,5X ,6, 6 ,7,12b-octahydro-2H-benzo wb xfuro w2,3-axquinazoline.2,4X-pyrimidin-2X-one; 81.3 Cirmmol. was from NENDuPont through DuMedical ŽStockholm, Sweden.; BDF8933 was a gift from Beiersdorf AG ŽHamburg, Germany.; BRL44408 was a gift from Smith, Kline and Beecham ŽEssex, UK.; spiroxatrine was from RBI ŽNatich, MA.. All other chemicals were from Sigma ŽSt. Louis, MO.. X
2.1. Animals and tissue preparations
2.3. a 2 A- and a 2 C -adrenoceptor autoradiographic procedure
Three adult male rats ŽSprague–Dawley, body weight 250–300 g., were killed by decapitation. The brains and spinal cords were rapidly removed, frozen in chilled isopentane Žy30 to y208C., and stored at y708C. Brain sections were cut with a cryostat Ž10 m m. and collected at the level of the anterior striatum Žbregma q1.2 to q0.7., while cervical spinal cord sections were collected at the C4–C5 level w18x. The slices were thaw-mounted on gelatin-coated glass slides, dried with a fan and stored at y708C.
For these studies, tissue sections were labeled with the 20-fold a 2C - to a 2A -adrenoceptor-selective antagonist w 3 HxMK912. Total binding was measured on ten slicesrtissue using 0.4 nM of w 3 HxMK912 without any competing drugs. Non-specific binding was measured on ten slicesrtissue in the presence of 2 m M of the nonspecific a 2-adrenoceptor antagonist BDF8933 w25x. In addition, two competition curves were constructed by including ten different concentrations of either the 80-fold a 2C to a 2A -selective drug spiroxatrine w24x or the 10-fold a 2A -
Fig. 1. Autoradiograms showing the labeling by 0.4 nM w 3 HxMK912 of a 2 -adrenoceptors in rat brain sections collected at the level of the anterior caudate putamen. A: total w 3 HxMK912 binding. B: non-specific binding obtained in the presence of 2 m M BDF8933. C: labeling of a 2C -adrenoceptors revealed by coincubation of w 3 HxMK912 and 185 nM of the a 2A -selective competing drug BRL44408. D: labeling of a 2A -adrenoceptors revealed by incubation of w 3 HxMK912 in the presence of 220 nM of the a 2C -selective competing drug spiroxatrine.
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to a 2C -selective drug BRL44408 w25x into the assays. Only one slide was used per competitor concentration, so for each set of experiments, 40 brain and 40 spinal cord slices were thawed and dried with a fan for 30 min and then preincubated in 50 mM Tris, 1.5 mM EDTA ŽpH 7.5. for 30 min. The slices were transferred to Tris-EDTA buffer containing 0.4 nM w 3 HxMK912 with or without competitors and incubated for 60 min at room temperature. After the incubation the slices were washed twice for 5 min in cold Ž48C. 50 mM Tris, 1.5 mM EDTA, then dipped in deionized water in order to remove buffer salts, and dried with a fan for 30 min. Labeled sections and plastic standards Ž 3 H-Microscales, 0.1–16 nCirmg; Amersham, Stockholm. were then placed in X-ray cassettes and exposed to radiation-sensitive film ŽAmersham Hyperfilm3 . H at y208C.
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weight based on the coexposed standards using NIH-Image software ŽNIH Image 1.54, NIMH, Bethesda, MD.. Brain regions selected for measurement ŽFig. 3. were the caudate putamen, the somatosensory frontoparietal cortex, the endopiriform nucleus and the septum w18x, and for the spinal cord the distal dorsal horn and the ventral horn w20x. Binding values obtained from the autoradiographic analysis were converted to pmolrmg tissue wet weight, based on the specific activity of the radioligand Žw 3 HxMK912.. The binding constants derived from the competition curves were calculated by fitting the data into the logistic four parameter function w29x using a curve fitting program written by us ŽWan System, Umea, ˚ Sweden.. Calculated Hill coefficients were in the range 0.83–1.32, but for clarity the Hill coefficients were set constant to unity in the curves displayed in Fig. 4.
2.4. DeÕelopment, quantification and analysis of results 3. Results After 5 weeks of exposure, the films were developed manually. The autoradiograms and standards were digitized with a video camera ŽCCD-72, Dage-MTI, Michigan City, IN.. Optical density was converted to nCirmg wet
Autoradiograms illustrating w 3 HxMK912 binding in striatal sections are shown in Fig. 1. High levels of total w 3 HxMK912 binding were obtained in cortical layers 1–4,
Fig. 2. Autoradiograms showing the labeling by 0.4 nM w 3 HxMK912 of a 2 -adrenoceptors in sections from the cervical spinal cord. A: total w 3 HxMK912 binding. B: non-specific binding obtained in the presence of 2 m M BDF8933. C: residual labeling in the presence of 185 nM of the a 2A -selective competing drug BRL44408. D: labeling of a 2A -adrenoceptors revealed by incubation of w 3 HxMK912 in the presence of 220 nM of the a 2C -selective competing drug spiroxatrine.
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the endopiriform nucleus, the claustrum, the septal nuclei, olfactory tubercle and shell region of the nucleus accumbens. Moderate binding was observed in the caudate putamen, cortical layers 5–6 and the core of the nucleus accumbens ŽFig. 1A.. In Fig. 1B is shown an autoradiogram demonstrating w 3 HxMK912 binding in the presence of 2 m M of the a 2-adrenoceptor antagonist BDF8933. As can be seen in Fig. 1B, very little w 3 HxMK912 remains bound in the presence of BDF8933, indicating that w 3 HxMK912 alone ŽFig. 1A. labeled almost only a 2-adrenoceptors. In the next set of experiments competition curves for either the a 2A -selective compound BRL44408 Žin the range 2 nM–45 m M. or the a 2C -selective compound spiroxatrine Žin the range 1 nM–18 m M. were constructed ŽFig. 1C,D.. Inspection of these autoradiograms revealed that spiroxatrine blocked w 3 HxMK912 binding most efficiently in the caudate putamen. In the caudate putamen most w 3 HxMK912 binding was blocked by 220 nM of spiroxatrine, a concentration that was virtually ineffective in the frontoparietal cortex, the endopiriform nucleus and the septum ŽFig. 1D.. By contrast, BRL44408 was most effective in competing for cortical, endopiriform and septal w 3 HxMK912 binding. In these tissues, 185 nM of BRL44408 blocked most of the w 3 HxMK912 binding, while the same concentration was virtually ineffective in the caudate nucleus ŽFig. 1C.. Exactly the same experimental design as described above for the striatal sections was also used for investigating the localization of a 2-adrenoceptor subtypes in sections of the cervical spinal cord. In Fig. 2 are shown representative autoradiograms of the results. Plain w 3 HxMK912 densely labeled the distal dorsal horn, while the remaining regions of spinal cord grey matter in the section were faintly labeled ŽFig. 2A.. In the presence of 2 m M of BDF8933 the section was almost blank indicating that w 3 HxMK912 specifically labeled a 2-adrenoceptors ŽFig. 2B.. In the presence of 185 nM BRL44408, the autoradiogram was very faint, with just some residual labeling in the distal dorsal horn ŽFig. 2C.. This indicates
Fig. 3. Schematic diagram of coronary brain sections derived from the atlas of Paxinos and Watson w18x, bregma q1.2 mm. Regions selected for measurement are the shadowed areas: somatosensory frontoparietal cortex ŽFrPaSS., endopiriform nucleus ŽEn., septum ŽS. and caudate putamen ŽCPu.. Each measurement was conducted in both halves of the brain.
Fig. 4. Representative competition curves, with IC 50 values inserted, for BRL44408 Žv . and spiroxatrine Ž'. for 0.4 nM w 3 HxMK912 binding in selected regions of rat brain and spinal cord. In A are shown competition curves for somatosensory frontoparietal cortex Žlayers 1–4., in B the endopiriform nucleus, in C the caudate putamen, and in D the distal dorsal horn. The data points were determined by measuring the optical densities of autoradiograms obtained from slices incubated with w 3 HxMK912 plus various concentrations of competitors. The ordinate shows w 3 HxMK912 binding in pmolesrmg wet weight of tissue, calculated on the basis of the coexposed 3 H-Microscales standards. Shown with arrows pointing to the right are the IC 50 values"S.E.M. for BRL44408, and with arrows pointing to the left the IC 50 values"S.E.M. for spiroxatrine.
that the main part of the a 2-adrenoceptors labeled by w 3 HxMK912 in the spinal cord represented the a 2A -subtype. Finally, in the presence of 220 nM of spiroxatrine, which preferentially blocks the a 2C -subtype, the autoradiogram ŽFig. 2D. looked fairly similar to the plain w 3 HxMK912 autoradiogram ŽFig. 2A.. The fact that it was fainter might be due to partial blockade of a 2A -adrenoceptors. Altogether, these results indicate that the a 2A -subtype predominates over the a 2C -subtype in the cervical spinal cord. The number of a 2-adrenoceptors labeled by 0.4 nM w 3 HxMK912 was quantified by comparing the optical densities of the autoradiograms labeled by 0.4 nM w 3 HxMK912 with the optical densities induced by the coexposed w 3 HxMicroscales standards. The measured brain areas are depicted in Fig. 3. The labeled optical densities Žfmolrmg wet weight, mean " S.E.M., n s 3, ten determinationsrexperiment. were estimated to be 25 " 1 for the caudate putamen, 28 " 3 for the somatosensory frontoparietal cortex, 61 " 4 for the endopiriform nucleus, and 66 " 6 for the septum. In the spinal cord the optical densities were 41 " 18 for the distal dorsal horn and 18 " 6 for the ventral horn. These latter areas were defined by a schematic sketch drawn previously w20x.
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The curves and IC 50 values obtained from the competition experiments with BRL44408 and spiroxatrine are shown in Fig. 4. The IC 50 values for the a 2A -selective drug BRL44408 ranged from 15 to 45 nM in all areas except for the caudate putamen where the IC 50 value was considerably higher Ž190 nM.. In the septum and ventral spinal cord, the two areas not shown in Fig. 4, the IC 50 values for BRL44408 were 21 " 9 nM and 17 " 12 nM, respectively Žmean " S.E.M., n s 3.. In the caudate putamen, the IC 50 value for the a 2C -selective drug spiroxatrine was low Ž24 nM., whereas in all other areas it was much higher, with values ranging from 85 to 180 nM. In the septum and ventral spinal cord Žnot shown in Fig. 4. the IC 50 values for spiroxatrine were 180 " 90 nM and 160 " 70 nM Žmean " S.E.M., n s 3.. For both drugs the Hill coefficients ranged from 0.83 to 1.32 indicating that none of the two competing drugs gave rise to any markedly biphasic curves in the measured areas. Since both spiroxatrine and BRL44408 are subtype selective this indicates that the majority of the sites labeled by w 3 HxMK912 in the caudate putamen were a 2C -adrenoceptors while the majority of the sites labeled in the other measured regions were a 2A -adrenoceptors.
4. Discussion In the present study, w 3 HxMK912 was used in combination with subtype-selective competitors to describe the distribution of striatal and cervical spinal cord a 2A - and a 2C -adrenoceptors using autoradiographic methods. The results showed that the a 2A -selective drug BRL44408 was more potent than the a 2C -selective drug spiroxatrine in competing for w 3 HxMK912 binding in the somatosensory cortex, endopiriform nucleus, septum, distal dorsal horn and ventral horn, whereas in the caudate putamen spiroxatrine was more potent than BRL44408. The results indicate that, e.g., the cortical, septal and dorsal spinal horn areas are enriched with a 2A -adrenoceptors, whereas a 2C -adrenoceptors were most abundant in the caudate putamen. In fact, at low concentrations of w 3 HxMK912, which shows 20-fold higher affinity for a 2C - as compared to a 2A -adrenoceptors w25x, the a 2C -adrenoceptors represent the vast majority of the labeled sites in the caudate putamen. In agreement with the present study are previous results showing that most regions of the rat central nervous system contain a 2A - and a 2C -adrenoceptors, but probably not a 2B -adrenoceptors w30x. Our results show a striking resemblance to the results obtained in a previous study where w 3 Hxrauwolscine, which is a 2C -selective in the rat w25x, was shown to label a 2-adrenoceptors preferentially in the caudate putamen, whereas w 3 Hxidazoxan preferentially labeled a 2-adrenoceptors in the septum and outer cerebral cortex w2x. In contrast to w 3 Hxrauwolscine, which is nonselective in the human, w 3 HxMK912 is a 2C -selective also in the human w26x. Thus, the results obtained from the
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present study provide a general strategy for differentiating between the a 2A - and a 2C -adrenoceptors using w 3 HxMK912 autoradiography. The physiological role of the a 2C -subtype of adrenoceptors is poorly understood. Therefore, we find it interesting that the level of a 2C -adrenoceptors was high in the rat striatum. Also present in the striatum are high numbers of b-adrenoceptors w19x. The presence of fairly large populations of a 2- and b-adrenoceptors in the striatum is somewhat puzzling, since the level of endogenous norepinephrine is comparatively low in this brain region, and the noradrenergic innervation of the caudate nucleus seems to be negligible Žsee Ref. w12x for references.. The precise functional role of norepinephrine in the striatum is far from clear. However, a complex relationship between norepinephrine and dopamine involving a facilitatory influence of the noradrenergic system on nigrostriatal dopaminergic neurotransmission has been suggested w12x. Regarding a 2-adrenoceptors, activation of the a 2A -subtype has been shown to inhibit the release of dopamine in the rabbit striatum w23x. In other brain regions, putative a 2C -adrenoceptors were shown to mediate inhibition of the synthesis of DOPA and norepinephrine w5x. In the present autoradiographic study a 2-adrenoceptors were visualized in the rat spinal cord. The highest density was present in the distal dorsal horn ŽFig. 2A.. This population seemed to represent mainly the a 2A -subtype, since most labeling was blocked by BRL44408 ŽFig. 2C., but not by spiroxatrine ŽFig. 2D.. However, using the present methods we were not able to specifically localize the a 2C -adrenoceptors, and we are not able to exclude the presence of low levels of a 2C -adrenoceptors in any specific area of the cervical spinal cord. In our previous radioligand binding study on rat spinal cord membranous homogenates w27x, the a 2C -subtype constituted about 5% and the a 2A -subtype 95% of the a 2-adrenoceptors in both the ventral and the dorsal halves of the spinal cord, indicating that the a 2C -adrenoceptors may be evenly distributed in the spinal cord. Based on in situ hybridization studies performed in rat it has been hypothesized that spinal a 2A -adrenoceptors, which are involved in modulating pain transmission, are localized postsynaptically on neurons located in the substantia gelatinosa. On the other hand, a 2C -adrenoceptors would be localized presynaptically on primary sensory neurons ŽC-afferents. w16x. As compared to the rat, in situ hybridization studies in the human spinal cord showed a different pattern of distribution for the a 2-adrenoceptor subtypes w22x. In the human spinal cord mRNAs encoding a 2A - and a 2B -adrenoceptors were widespread, whereas mRNAs encoding a 2C -adrenoceptors were detected only in the lumbar spinal cord. These results indicate that large species variations exist in the distribution of a 2-adrenoceptor subtypes. In summary, using w 3 HxMK912 in combination with the a 2A -selective drug BRL44408 or the a 2C -selective drug spiroxatrine, a 2A - and a 2C -adrenoceptors were visualized
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autoradiographically in rat nervous tissue. Relatively high numbers of a 2C -adrenoceptors were detected in the striatum, while predominantly a 2A -adrenoceptors were detected in, e.g., the cerebral cortex, spinal cord, endopiriform nucleus and septum. The approach used in the present study provides a method for the selective identification of a 2A - and a 2C -adrenoceptors in the CNS.
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Acknowledgements Supported by the Swedish MRC 04X-05957, the Berth von Kantzow, the Magnus Bergwall, the Clas Groschinsky ˚ Wiberg Foundations. and the Ake
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Research report
Autoradiographic studies of central a 2A- and a 2C-adrenoceptors in the rat using w 3 HxMK912 and subtype-selective drugs Staffan Uhlen ´ a
a, )
, Jonas Lindblom a , Allan Johnson b , Jarl E.S. Wikberg
a
Department of Pharmaceutical Biosciences, DiÕision of Pharmacology, Box 591, Uppsala UniÕersity, S-751 24 Uppsala, Sweden b Department of Psychiatry, Uppsala UniÕersity, Uppsala, Sweden Accepted 1 July 1997
Abstract In the present study we examined the distribution of a 2A- and a 2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between a 2A- and a 2C-adrenoceptors, the slices were incubated with w 3 HxMK912 in the presence of graded concentrations of the a 2A-selective drug, BRL44408, or the a 2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM w 3 HxMK912 plus 185 nM BRL44408 selectively labeled a 2C-adrenoceptors, while 0.4 nM w 3 HxMK912 plus 220 nM spiroxatrine selectively labeled a 2A-adrenoceptors. Using this approach, a 2C-adrenoceptors were detected in the striatum, while a 2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus. q 1997 Elsevier Science B.V. Keywords: Autoradiography; a 2A -Adrenoceptor; a 2C -Adrenoceptor; w 3 HxMK912; Spinal cord; Striatum
1. Introduction Based on structural and pharmacological evidence, the a 2-adrenoceptors are currently classified into three subtypes designated a 2A -, a 2B - and a 2C -adrenoceptors w3x. The rat and the human orthologs of the a 2A -adrenoceptor show somewhat discrepant pharmacological properties, and sometimes the rat ortholog is designated a 2D . Still the consensus of today is that there exist only three a 2-adrenoceptor subtypes within one species w9x. In radioligand binding studies performed on membranous homogenates, and using the 20-fold a 2C - to a 2A selective radioligand w 3 HxMK912, both a 2A - and a 2C adrenoceptors were detected in the rat cerebral cortex w25x, striatum, and at different levels of the spinal cord w27x. Similarly, a 2A - and a 2C -adrenoceptors were detected in membranes from the human caudate nucleus using either w 3 Hxyohimbine w17x or w 3 HxRX821002 w21x. A more precise anatomical localization of a 2-adrenoceptors has been performed in nervous tissue using autoradiography. Thus, a 2-adrenoceptors were visualized in different sections of the rat brain and spinal cord using
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Corresponding author. Fax: q46 Ž18 . 559-718; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 6 0 - 3
w 3 H x p-aminoclonidine w28x, w 3 H xrauwolscine w2,20x, w 3 Hxidazoxan w2x and w 3 Hxrilmenidine w10x. Autoradiographic studies with w 3 Hxrauwolscine, w 3 Hxyohimbine and w 3 HxRX821002 have given further information on the distribution of a 2-adrenoceptors in the human brain and spinal cord w4,8,17x. However, in none of these autoradiographic studies any attempt was made to characterize which a 2-adrenoceptor subtypes became labeled. Northern blot analysis indicates that mRNAs encoding the a 2A - and a 2C -adrenoceptors were widely distributed in the rat CNS whereas a 2B -encoding mRNA was detectable only in the diencephalon w30x. Moreover, using in situ hybridization it was shown that mRNAs encoding both a 2A - and a 2C -adrenoceptors are present in the cerebral cortex, cerebellum, spinal cord, and other parts of the rat nervous system w14–16x. Drugs active on a 2-adrenoceptors are in clinical use, or may find clinical use in the near future, as antidepressants, for the induction of anesthesia or analgesia, for the treatment of glaucoma Žsee w7,11x., for the treatment of attention-deficit hyperactivity disorders w1x, for treatment of hypertension w13x, and in the field of Parkinson’s disease w6,23x. Characterization of the specific a 2-adrenoceptor subtypes involved in the different physiological effects mediated through the a 2-adrenoceptor seems warranted. The present study was undertaken to develop an ap-
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proach for the delineation of a 2A - and a 2C -adrenoceptors in the rat central nervous system using receptor autoradiographic techniques. Our results with w 3 HxMK912 in conjunction with subtype-selective drugs show that a 2C adrenoceptors are detectable in the striatum, while a 2A adrenoceptors are detectable in the somatosensory frontoparietal cerebral cortex, the endopiriform nucleus, the septum and the spinal cord distal dorsal horn and ventral horn. 2. Materials and methods
2.2. Radioligands, drugs and chemicals w 3 HxMK912 ŽŽ2 S,12bS .-1X ,3X-dimethylspiroŽ1,3,4,5X ,6, 6 ,7,12b-octahydro-2H-benzo wb xfuro w2,3-axquinazoline.2,4X-pyrimidin-2X-one; 81.3 Cirmmol. was from NENDuPont through DuMedical ŽStockholm, Sweden.; BDF8933 was a gift from Beiersdorf AG ŽHamburg, Germany.; BRL44408 was a gift from Smith, Kline and Beecham ŽEssex, UK.; spiroxatrine was from RBI ŽNatich, MA.. All other chemicals were from Sigma ŽSt. Louis, MO.. X
2.1. Animals and tissue preparations
2.3. a 2 A- and a 2 C -adrenoceptor autoradiographic procedure
Three adult male rats ŽSprague–Dawley, body weight 250–300 g., were killed by decapitation. The brains and spinal cords were rapidly removed, frozen in chilled isopentane Žy30 to y208C., and stored at y708C. Brain sections were cut with a cryostat Ž10 m m. and collected at the level of the anterior striatum Žbregma q1.2 to q0.7., while cervical spinal cord sections were collected at the C4–C5 level w18x. The slices were thaw-mounted on gelatin-coated glass slides, dried with a fan and stored at y708C.
For these studies, tissue sections were labeled with the 20-fold a 2C - to a 2A -adrenoceptor-selective antagonist w 3 HxMK912. Total binding was measured on ten slicesrtissue using 0.4 nM of w 3 HxMK912 without any competing drugs. Non-specific binding was measured on ten slicesrtissue in the presence of 2 m M of the nonspecific a 2-adrenoceptor antagonist BDF8933 w25x. In addition, two competition curves were constructed by including ten different concentrations of either the 80-fold a 2C to a 2A -selective drug spiroxatrine w24x or the 10-fold a 2A -
Fig. 1. Autoradiograms showing the labeling by 0.4 nM w 3 HxMK912 of a 2 -adrenoceptors in rat brain sections collected at the level of the anterior caudate putamen. A: total w 3 HxMK912 binding. B: non-specific binding obtained in the presence of 2 m M BDF8933. C: labeling of a 2C -adrenoceptors revealed by coincubation of w 3 HxMK912 and 185 nM of the a 2A -selective competing drug BRL44408. D: labeling of a 2A -adrenoceptors revealed by incubation of w 3 HxMK912 in the presence of 220 nM of the a 2C -selective competing drug spiroxatrine.
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to a 2C -selective drug BRL44408 w25x into the assays. Only one slide was used per competitor concentration, so for each set of experiments, 40 brain and 40 spinal cord slices were thawed and dried with a fan for 30 min and then preincubated in 50 mM Tris, 1.5 mM EDTA ŽpH 7.5. for 30 min. The slices were transferred to Tris-EDTA buffer containing 0.4 nM w 3 HxMK912 with or without competitors and incubated for 60 min at room temperature. After the incubation the slices were washed twice for 5 min in cold Ž48C. 50 mM Tris, 1.5 mM EDTA, then dipped in deionized water in order to remove buffer salts, and dried with a fan for 30 min. Labeled sections and plastic standards Ž 3 H-Microscales, 0.1–16 nCirmg; Amersham, Stockholm. were then placed in X-ray cassettes and exposed to radiation-sensitive film ŽAmersham Hyperfilm3 . H at y208C.
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weight based on the coexposed standards using NIH-Image software ŽNIH Image 1.54, NIMH, Bethesda, MD.. Brain regions selected for measurement ŽFig. 3. were the caudate putamen, the somatosensory frontoparietal cortex, the endopiriform nucleus and the septum w18x, and for the spinal cord the distal dorsal horn and the ventral horn w20x. Binding values obtained from the autoradiographic analysis were converted to pmolrmg tissue wet weight, based on the specific activity of the radioligand Žw 3 HxMK912.. The binding constants derived from the competition curves were calculated by fitting the data into the logistic four parameter function w29x using a curve fitting program written by us ŽWan System, Umea, ˚ Sweden.. Calculated Hill coefficients were in the range 0.83–1.32, but for clarity the Hill coefficients were set constant to unity in the curves displayed in Fig. 4.
2.4. DeÕelopment, quantification and analysis of results 3. Results After 5 weeks of exposure, the films were developed manually. The autoradiograms and standards were digitized with a video camera ŽCCD-72, Dage-MTI, Michigan City, IN.. Optical density was converted to nCirmg wet
Autoradiograms illustrating w 3 HxMK912 binding in striatal sections are shown in Fig. 1. High levels of total w 3 HxMK912 binding were obtained in cortical layers 1–4,
Fig. 2. Autoradiograms showing the labeling by 0.4 nM w 3 HxMK912 of a 2 -adrenoceptors in sections from the cervical spinal cord. A: total w 3 HxMK912 binding. B: non-specific binding obtained in the presence of 2 m M BDF8933. C: residual labeling in the presence of 185 nM of the a 2A -selective competing drug BRL44408. D: labeling of a 2A -adrenoceptors revealed by incubation of w 3 HxMK912 in the presence of 220 nM of the a 2C -selective competing drug spiroxatrine.
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the endopiriform nucleus, the claustrum, the septal nuclei, olfactory tubercle and shell region of the nucleus accumbens. Moderate binding was observed in the caudate putamen, cortical layers 5–6 and the core of the nucleus accumbens ŽFig. 1A.. In Fig. 1B is shown an autoradiogram demonstrating w 3 HxMK912 binding in the presence of 2 m M of the a 2-adrenoceptor antagonist BDF8933. As can be seen in Fig. 1B, very little w 3 HxMK912 remains bound in the presence of BDF8933, indicating that w 3 HxMK912 alone ŽFig. 1A. labeled almost only a 2-adrenoceptors. In the next set of experiments competition curves for either the a 2A -selective compound BRL44408 Žin the range 2 nM–45 m M. or the a 2C -selective compound spiroxatrine Žin the range 1 nM–18 m M. were constructed ŽFig. 1C,D.. Inspection of these autoradiograms revealed that spiroxatrine blocked w 3 HxMK912 binding most efficiently in the caudate putamen. In the caudate putamen most w 3 HxMK912 binding was blocked by 220 nM of spiroxatrine, a concentration that was virtually ineffective in the frontoparietal cortex, the endopiriform nucleus and the septum ŽFig. 1D.. By contrast, BRL44408 was most effective in competing for cortical, endopiriform and septal w 3 HxMK912 binding. In these tissues, 185 nM of BRL44408 blocked most of the w 3 HxMK912 binding, while the same concentration was virtually ineffective in the caudate nucleus ŽFig. 1C.. Exactly the same experimental design as described above for the striatal sections was also used for investigating the localization of a 2-adrenoceptor subtypes in sections of the cervical spinal cord. In Fig. 2 are shown representative autoradiograms of the results. Plain w 3 HxMK912 densely labeled the distal dorsal horn, while the remaining regions of spinal cord grey matter in the section were faintly labeled ŽFig. 2A.. In the presence of 2 m M of BDF8933 the section was almost blank indicating that w 3 HxMK912 specifically labeled a 2-adrenoceptors ŽFig. 2B.. In the presence of 185 nM BRL44408, the autoradiogram was very faint, with just some residual labeling in the distal dorsal horn ŽFig. 2C.. This indicates
Fig. 3. Schematic diagram of coronary brain sections derived from the atlas of Paxinos and Watson w18x, bregma q1.2 mm. Regions selected for measurement are the shadowed areas: somatosensory frontoparietal cortex ŽFrPaSS., endopiriform nucleus ŽEn., septum ŽS. and caudate putamen ŽCPu.. Each measurement was conducted in both halves of the brain.
Fig. 4. Representative competition curves, with IC 50 values inserted, for BRL44408 Žv . and spiroxatrine Ž'. for 0.4 nM w 3 HxMK912 binding in selected regions of rat brain and spinal cord. In A are shown competition curves for somatosensory frontoparietal cortex Žlayers 1–4., in B the endopiriform nucleus, in C the caudate putamen, and in D the distal dorsal horn. The data points were determined by measuring the optical densities of autoradiograms obtained from slices incubated with w 3 HxMK912 plus various concentrations of competitors. The ordinate shows w 3 HxMK912 binding in pmolesrmg wet weight of tissue, calculated on the basis of the coexposed 3 H-Microscales standards. Shown with arrows pointing to the right are the IC 50 values"S.E.M. for BRL44408, and with arrows pointing to the left the IC 50 values"S.E.M. for spiroxatrine.
that the main part of the a 2-adrenoceptors labeled by w 3 HxMK912 in the spinal cord represented the a 2A -subtype. Finally, in the presence of 220 nM of spiroxatrine, which preferentially blocks the a 2C -subtype, the autoradiogram ŽFig. 2D. looked fairly similar to the plain w 3 HxMK912 autoradiogram ŽFig. 2A.. The fact that it was fainter might be due to partial blockade of a 2A -adrenoceptors. Altogether, these results indicate that the a 2A -subtype predominates over the a 2C -subtype in the cervical spinal cord. The number of a 2-adrenoceptors labeled by 0.4 nM w 3 HxMK912 was quantified by comparing the optical densities of the autoradiograms labeled by 0.4 nM w 3 HxMK912 with the optical densities induced by the coexposed w 3 HxMicroscales standards. The measured brain areas are depicted in Fig. 3. The labeled optical densities Žfmolrmg wet weight, mean " S.E.M., n s 3, ten determinationsrexperiment. were estimated to be 25 " 1 for the caudate putamen, 28 " 3 for the somatosensory frontoparietal cortex, 61 " 4 for the endopiriform nucleus, and 66 " 6 for the septum. In the spinal cord the optical densities were 41 " 18 for the distal dorsal horn and 18 " 6 for the ventral horn. These latter areas were defined by a schematic sketch drawn previously w20x.
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The curves and IC 50 values obtained from the competition experiments with BRL44408 and spiroxatrine are shown in Fig. 4. The IC 50 values for the a 2A -selective drug BRL44408 ranged from 15 to 45 nM in all areas except for the caudate putamen where the IC 50 value was considerably higher Ž190 nM.. In the septum and ventral spinal cord, the two areas not shown in Fig. 4, the IC 50 values for BRL44408 were 21 " 9 nM and 17 " 12 nM, respectively Žmean " S.E.M., n s 3.. In the caudate putamen, the IC 50 value for the a 2C -selective drug spiroxatrine was low Ž24 nM., whereas in all other areas it was much higher, with values ranging from 85 to 180 nM. In the septum and ventral spinal cord Žnot shown in Fig. 4. the IC 50 values for spiroxatrine were 180 " 90 nM and 160 " 70 nM Žmean " S.E.M., n s 3.. For both drugs the Hill coefficients ranged from 0.83 to 1.32 indicating that none of the two competing drugs gave rise to any markedly biphasic curves in the measured areas. Since both spiroxatrine and BRL44408 are subtype selective this indicates that the majority of the sites labeled by w 3 HxMK912 in the caudate putamen were a 2C -adrenoceptors while the majority of the sites labeled in the other measured regions were a 2A -adrenoceptors.
4. Discussion In the present study, w 3 HxMK912 was used in combination with subtype-selective competitors to describe the distribution of striatal and cervical spinal cord a 2A - and a 2C -adrenoceptors using autoradiographic methods. The results showed that the a 2A -selective drug BRL44408 was more potent than the a 2C -selective drug spiroxatrine in competing for w 3 HxMK912 binding in the somatosensory cortex, endopiriform nucleus, septum, distal dorsal horn and ventral horn, whereas in the caudate putamen spiroxatrine was more potent than BRL44408. The results indicate that, e.g., the cortical, septal and dorsal spinal horn areas are enriched with a 2A -adrenoceptors, whereas a 2C -adrenoceptors were most abundant in the caudate putamen. In fact, at low concentrations of w 3 HxMK912, which shows 20-fold higher affinity for a 2C - as compared to a 2A -adrenoceptors w25x, the a 2C -adrenoceptors represent the vast majority of the labeled sites in the caudate putamen. In agreement with the present study are previous results showing that most regions of the rat central nervous system contain a 2A - and a 2C -adrenoceptors, but probably not a 2B -adrenoceptors w30x. Our results show a striking resemblance to the results obtained in a previous study where w 3 Hxrauwolscine, which is a 2C -selective in the rat w25x, was shown to label a 2-adrenoceptors preferentially in the caudate putamen, whereas w 3 Hxidazoxan preferentially labeled a 2-adrenoceptors in the septum and outer cerebral cortex w2x. In contrast to w 3 Hxrauwolscine, which is nonselective in the human, w 3 HxMK912 is a 2C -selective also in the human w26x. Thus, the results obtained from the
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present study provide a general strategy for differentiating between the a 2A - and a 2C -adrenoceptors using w 3 HxMK912 autoradiography. The physiological role of the a 2C -subtype of adrenoceptors is poorly understood. Therefore, we find it interesting that the level of a 2C -adrenoceptors was high in the rat striatum. Also present in the striatum are high numbers of b-adrenoceptors w19x. The presence of fairly large populations of a 2- and b-adrenoceptors in the striatum is somewhat puzzling, since the level of endogenous norepinephrine is comparatively low in this brain region, and the noradrenergic innervation of the caudate nucleus seems to be negligible Žsee Ref. w12x for references.. The precise functional role of norepinephrine in the striatum is far from clear. However, a complex relationship between norepinephrine and dopamine involving a facilitatory influence of the noradrenergic system on nigrostriatal dopaminergic neurotransmission has been suggested w12x. Regarding a 2-adrenoceptors, activation of the a 2A -subtype has been shown to inhibit the release of dopamine in the rabbit striatum w23x. In other brain regions, putative a 2C -adrenoceptors were shown to mediate inhibition of the synthesis of DOPA and norepinephrine w5x. In the present autoradiographic study a 2-adrenoceptors were visualized in the rat spinal cord. The highest density was present in the distal dorsal horn ŽFig. 2A.. This population seemed to represent mainly the a 2A -subtype, since most labeling was blocked by BRL44408 ŽFig. 2C., but not by spiroxatrine ŽFig. 2D.. However, using the present methods we were not able to specifically localize the a 2C -adrenoceptors, and we are not able to exclude the presence of low levels of a 2C -adrenoceptors in any specific area of the cervical spinal cord. In our previous radioligand binding study on rat spinal cord membranous homogenates w27x, the a 2C -subtype constituted about 5% and the a 2A -subtype 95% of the a 2-adrenoceptors in both the ventral and the dorsal halves of the spinal cord, indicating that the a 2C -adrenoceptors may be evenly distributed in the spinal cord. Based on in situ hybridization studies performed in rat it has been hypothesized that spinal a 2A -adrenoceptors, which are involved in modulating pain transmission, are localized postsynaptically on neurons located in the substantia gelatinosa. On the other hand, a 2C -adrenoceptors would be localized presynaptically on primary sensory neurons ŽC-afferents. w16x. As compared to the rat, in situ hybridization studies in the human spinal cord showed a different pattern of distribution for the a 2-adrenoceptor subtypes w22x. In the human spinal cord mRNAs encoding a 2A - and a 2B -adrenoceptors were widespread, whereas mRNAs encoding a 2C -adrenoceptors were detected only in the lumbar spinal cord. These results indicate that large species variations exist in the distribution of a 2-adrenoceptor subtypes. In summary, using w 3 HxMK912 in combination with the a 2A -selective drug BRL44408 or the a 2C -selective drug spiroxatrine, a 2A - and a 2C -adrenoceptors were visualized
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autoradiographically in rat nervous tissue. Relatively high numbers of a 2C -adrenoceptors were detected in the striatum, while predominantly a 2A -adrenoceptors were detected in, e.g., the cerebral cortex, spinal cord, endopiriform nucleus and septum. The approach used in the present study provides a method for the selective identification of a 2A - and a 2C -adrenoceptors in the CNS.
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Acknowledgements Supported by the Swedish MRC 04X-05957, the Berth von Kantzow, the Magnus Bergwall, the Clas Groschinsky ˚ Wiberg Foundations. and the Ake
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