Molecular Brain Research, 12 (1992) 293-314 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0169-328X/92/$05.00

293

BRESM 70380

Region-specific expression of G A B A A receptor tx3 and a 4 subunits m R N A s in the rat brain T. Araki and M. Tohyama Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka (Japan) (Accepted 27 August 1991)

Key words: GABAA receptor a 3 subunit; GABA A receptor a 4 subunit; Cellular localization; Rat brain; In situ hybridization histochemistry

The expression of m R N A s encoding the a 3 and a 4 subunits of the 7-aminobutyr£c acid A (GABAA) receptor in the rat brain was investigated by in situ hybridization histochemistry. Both subunits showed a wide but uneven distribution, which did not coincide with the distribution of any other subunit so far reported. The cerebral cortex, anterior olfactory nucleus, lateral septum, subicuhm, lateral and medial nuclei of the amygdaloid complex, anterior nuclei of the thalamus, pars compacta of the substantia nigra, trigeminal sensory nuclei, and cochlear nucleus were some of the areas where strong expression of mRNA for both the a 3 and a 4 subunits was detected. In the mitral cell layer of the olfactory bulb, the preoptic area and locus coeruleus, strong expression of only the a a subonit was detected. In the granular cell layer of the olfactory bulb, caudate-pummen, tonia tecta, pyramidal cell layer of the CA region and granular cell layer of the dentate gyms in the hippocampal formation, dorsomediai and ventrolateral nuclei of the thalamus, dorsal part of the lateral geniculate body, prcofivary nuclei and pontine nuclei, only the a 4 subunit showed strong expression. The diverse distribution of these two subunits is considered to indicate that each has a different role in the central nervous system. INTRODUCTION 7-Aminobutyric acid ( G A B A ) is considered to be one of the most abundant inhibitory neurotransmitters in the mammalian central nervous system 2°'22. Biochemical and electrophysinlogical studies have shown that there are at least two types of G A B A receptor in the central nervous system, namely, the G A B A A and G A B A B receptors. The G A B A A receptor is a m e m b e r o f the ligand-gated ion channel family of receptors and has been shown to have a heterooligomeric structure comprising a number of subunits and variants (al_~, fll-3, •1-2, and 6) 7-9'

a few autoradiography plates, so the overall distribution of the cells expressing these subunit m R N A s is still obscure. In addition, the identification of nuclei is difficult when using film autoradiography. In the present study, we used synthesized antisense oligonucleotide probes for the a3 and a4 subunit m R N A s of G A B A A receptor to examine the detailed localization of cells containing these m R N A s in the rat brain. Our results were then compared with those of mapping studies of other subunits of the G A B A A receptor which have already been reported.

22,23,27,2a

MATERIALS AND METHODS

Using in situ hybridization histochemistry, we have recently demonstrated that fl~-3 subunit m R N A s showed region-specific expression in the rat brain 3°. In addition, Wisden et al. compared the localization of cells containing a t - a 3 subunit m R N A s in the cerebral cortex and concluded that these subuniis also showed region-specific expression in the central nervous system 26. With regard to the localization of cells containing a subunit m R N A s , detailed m a p p i n g of the rat brain has been performed for the at subunit 6'29, but for the a2_ 4 subunit m R N A s , the cellular localization has only b e e n determined in the cerebral cortex, A m m o n ' s horn, the dentate gyms, and the cerebellum t2'26. Moreover, these studies presented only

Animals and tissue preparations Young male Wistar rats (postnatal day 14) were used. They were anesthetized with pentobarbital (50 mg/kg, i.p.) and killed by decapitation. The brains were quickly removed and frozen with powdered dry ice. Sections 10-15 /~m thick were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and stored at -80°C until hybridization (up to I month). in situ hybridization The hybridization procedure was essentially the same as that described by Block et al.4. Frozen sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 5 min at room temperature. (Unless specified otherwise, the following procedures were all carried out at room temperature.) They were rinsed 3 times (5 sin each) in 4 x SSC (1 x SSC contained 0.15 M sodium chloride and 0.015 M sodium citrate), immersed for 1 h in 4 x SSC containing 1 ~< Denh~rdt's solution (1 x Denhardt's solution con-

Correspondence: T. Araki, Department of Anatomy and Neuroscience, Osaka University Ivledical School, 2-2 Yamadaoka, Suita, 565, Japan.

294 rained 0.02% polyvinylpyroridone K-30, 0.02% bovine serum albumin, and 0.02% Ficoil 400), and dehydrated with a graded ethanol series. The sections were treated with chloroform for I0 miu to remove fat from the tissue, and then immersed in 100% ethanol twice for 5 rain each before hybridization. Hybridization was performed by incubating the sections with a buffer (4 × SSC, 50% deionized fomamide, 0.12 M phosphate buffer (pH 7.2), 1 × Denhardt's solution, 2.5% tRNA, 10% dextran sulfate, 50 mM dithiothreitol) containing [a-35S]dATP (10000-15000 Ci/mmol (37-55.5 TBq/mmol)) labeled probe (6-9 × 106dpm/ml)for 24-48 h at 41°(?. After hybridization the sections were rinsed in 4 × SSC (pH 7.0) for 10 min, and then rinsed three times in 1 × SSC at 65°C for 20 min each time. Then the sections were dehydrated with a graded ethanol series (70-100%) and coated with Ilford K-5 emulsion (diluted l:l with water). These sections were exposed for 4--6 weeks in a tightly sealed dark box at 4°C. After being developed in D-19 developer, fixed wR~lphotographic fixer and washed with tap water, the sections were cou~te~tained wP.h thionin solution to allow morphological identification of the central nervous system nuclei.

Oligonucleotideprobes The oligonucleotide probes (each 48 met) were synthesized using an Applied Biosystems DNA synthesizer and then purified by high pressure liquid chromatography (ODS column chromatography). The probes consisted of antisense nucleotides and were derived from rat DNA sequences complimentary to those of the GABAA receptor a3 and 6t4 subunits: complimentary to bases 1545-1592 for the a 3 probe and to bases 1279-1326 for the a4 probe 13. A DNA homology search showed that our probes shared 52.08% identity with each other and less than 54.17% identity with the cDNA sequences for other subtypes of the GABAA receptor.

Specificity and corttrols The specificity of the hybridization signals was confirmed a.~ follows. 1. No hybridization signal was detected in a competition experiment involving prehybridization of sections with excess unlabeled probe. 2. The a 3 signals were not affected when hybridization was performed with an excess of the unlabeled a 4 probe, while the a 4 signals were not affected by hybridized with an excess of the unlabeled a 3 probe. 3. Prior to in-situ hybridization, sections were incubated for 1 h at 37°C with a buffer containing 10 mM Tris-HCI (pH 7.5), 1 mM ethylendi~minetetraaceticacid and pancreatic RNase A (10/~M/ml, Sigma): no hybridization signal was detected after such treatment. (1-3 : data not shown).

Nomenclature Terminology was based on the atlas of Paxinos and Watson~s.

RESULTS To identify the neurons with positive signals, the grain densities of the neurons in some brain areas and the background densities of the sections were determined for our probes a~ reported elsewhere tS. Then the signal-tonoise (S/N) ratio was calculated for each probe. In the present study, neurons with a grain density at least 3 times higher than the background density were consid. ered to be positive. The positive neurons were divided into 3 categories according to their S/N ratio, i.e. strongly labeled neurons (S/N ratio > 8), moderately labeled neurons (S/N ratio 5-8), and weakly labeled neurons (S/N ratio 3-5).

Olfactory bulb and its related areas Main olfactory bulb (Figs. la and 2a, b) Both the a3 and a4 subunits of the GABAA receptor were distributed in t.he olfactory bulb, with a 3 subunit m R N A being strongly expressed by almost all the cells in the mitral cell layer. The external plexiform layer contained strongly positive cells that were distributed mainly in its outer part. The a4 subunit m R N A was expressed moderately in the external plexiform later and the glomerular layer, but not in the mitral cell layer.

Accessory olfactory bulb (Figs. la and 2a, b) In the accessory olfactory nucleus, the a 3 subunit was strongly expressed by almost all the cells in the mitral cell layer, while no a4-positive cells were observed.

Anterior olfactory nucleus (Figs. lb and 2c, d) All the subnuclei in the anterior olfactory nucleus strongly expressed as and a4 subunits and the signal intensity of each nucleus did not vary markedly. No labeled neurons were observed in the lateral olfactory tract.

Piriform cortex (Figs. lc-i and 3c, d) Most of the cells in layer 2 of the piriform cortex were labeled moderately by the as probe, and strongly by the a4 probe, a s subunit expression was stronger in the rostral area and weak or absent in caudal area. However, signals for the a4 subunit showed no difference in intensity between the caudal to rostral areas. In layer 1 and 3, only a few positive neurons were found.

Cerebral cortex Isocortex (Figs. l b - m and 3a, b) Both the a3 and a4 subunits were expressed in layers I I - V I of the isocortex, although their distribution differed. The a 3 subunit showed the strongest expression in the layers V and VI, while neurons labeled by the a4 probe showed a similar intensity throughout layers I I VI. The distribution pattern and density or intensity of both as- and a4-positive cells were similar throughout all the subregions of the isocortex. Both pyramidal cells and non-pyramidal cells were labeled by the two probes.

Allocortex (anterior cingulate cortex, retrosplenial cortex, perirhinal cortex) (Figs. lc-m) All the subregions of the allocortex expressed both the as and a4 subunits strongly. The signal intensity for both subunits in all the allocortex was almost the same as the strong signal in the isocortex, and the positive cells were evenly distributed.

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310 expression of mRNA for 54 subunit was moderate to weak in this region.

General somatosensory system (Fig. In-t) Both subunits were moderately expressed in the gracil and cuneate nuclei. The pars caudalis, interpositus and oralis of the spinal trigeminal nucleus strongly expressed both subunits. Positive cells were distributed both in the magnocellular part of this nucleus and in the substantia gelatinosa. The signal intensity of labeling for the two subunits were almost the same in these areas. However, the mesencephalic trigeminal nucleus shared rather weaker labeling by the 53 probe, compared to the other trigeminal subnuclei, althou~ a4-positive signals were not reduced. Special somatosensory system Vestibular system (Figs. 1p-r) Moderately 53-positive cells were observed in the vestibular nucleus and there was no variation of signal intensity throughout the subnuclei. The a4-positive signals ;;,ere strong in the medial vestibular subnuclei and adjacent areas, while other region expressed a4-positive signal moderately. Auditory system (Figs. l m - q and 8e,f) In both the ventral and dorsal pa~s of the cechlear nucleus, superior olivary nucleus, ventral preolivary nucleus, and trapezoid nucleus strongly expressed a4 subunit. The lateral lemn~scus and inferior colliculus moderately expressed a4 subunit. Expression of the mRNA for a 3 subunit was moderate in all of the above-mentioned areas, with the exception of the inferior colliculus which expressed scattered weakly labeled neurons. Visual system (Figs. 1k-m) Cells moderately positive for 53 subunit were desseminated in each layer of the superior colliculus. Expression of the 54 subunit was strong to moderate in this region. Strong expression for 54 subunit was mainly observed in the superficial gray layer and the intermediate gray layer. In the parabigeminal nucleus, a3 subunit was weakly expressed, while 54 ~ubunit was moderately expressed. General somatomotor system and branchiomotor system (Fig. lp-t) Motor neurons in the cranial motor nuclei innervating striated muscle moderately expressed both subunits. General visceromotor system (Fig. lk, n) The dorsal motor nucleus of the vagus and t h e Edinger-Westphal nucleus moderately expressed both of the two subunits. Reticular formation (Fig. In-t) The reticular formation generally showed weakly positive signals for the a 3 subunit irrespective of the level in

the brain. Some of the large neurons in the medial twothirds of the reticular formation and parvocellular reticular nucleus moderately expressed a3 subunit. Expression of the mRNA for a4 subunit was distributed widely in almost all the regions of the reticular formation. The labeling intensity of the a4-positive cells was moderate. Reticulotegrnental nucleus of pons showed strong expression of a 4 subunit, while it lacked a3-positive cells.

Raphe system (Fig. lm-O The dorsal raphe nucleus strongly expressed 53 subunit, but it expressed a4 subunit weakly to moderately. Other nuclei moderately expressed both subunits in general. Laterodorsal tegmentum of the pons (Figs. ln,o and 8c, d) Neurons in the laterodorsal tegmental nucleus only weakly expressed both subunits. The a3 subunit was expressed strongly in the locus coeruleus, while rt4 subunit was moderately expressed. Expression of mRNA of 53 subunit was strong in the parabrachial nucleus, though that of 54 subunit was moderate. Other lower brainstem nuclei (Fig. ll, m,o-O Both the a 3 and 54 subunits generally showed moderate expression in other lower brainstem nuclei, with the exception of the inferior olivary nucleus which contained densely packed cells showing strong expression of the both subunits. The pontine nucleus strongly expressed 54 subunit (Fig. 8a,b). Cerebellum Both the aa and a4 subunit were moderately to strongly expressed in the molecular layer of the cerebellum. Purkinje cell layer and the granular cell layer lacked positive signals for the two subunits. In the cerebellar nuclei, the a3 subunit was moderately expressed, but expression of 54 subunit was weak.

DISCUSSION

Comparison with previous reports on the localization of a z and a 4 subunit mRNAs in the brain Using it. situ hybridization histochemistry at the microscopic level, Wisden et al. reported that a3 subunit mRNA was expressed in the deep layers of the cerebral cortex, the s,lbiculum, the pyramidal cells of Ammon's horn, and the granular cell layer of the dentate gyrus26. In addition, Malherbe et al. have recently shown that aa subunit mRNA was expressed widely in brain such as the olfactory bulb (mitral cell layer), cerebral cortex, pyra-

311 midal cell layer of Ammon's horn, granular cell layer of the dentate gyms, the thalamus, the midbrain, and the cerebellumTM. However, in their study, the exact cellular localization of the a3 subunit mRNA was unclear because they used film antoradiographic techniques. The present study confirmed that cells expressing a 3 subunit mRNA were widely distributed in the brain and further elucidated their detailed cellular localization. Many sites were newly identified as containing neurons expressing a3 subunit mRNA, such as the lateral and medial septal nuclei, various thalamic and hypothalamic nuclei, the piriform cortex, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, the central gray matter of the midbrain, the vestibular nuclei, the locus coeruleus, the parabrachiai area, the trigeminal sensory nuclei and nucleus of the tractus solltarius etc. The location of the a4 subunit mRNA in the brain has also been examined by a few authors. Khrestchatisky et al. showed that all the cortical layers and the pyramidal cells of Ammon's horn expressed a4 subunit, but it did not detected in other brain areas7. Malherbe et al. examined the localization of as subunit mRNA in the rat brain using film autoradiographyTM. Since the complete sequence of a~ subunit cDNA is identical to that of a4 subunit cDlqA, the sites labeled by their probe appeared to represent those containing a4 subunit mRNA. They reported that the following sites were labeled (though as mentioned above, the exact cellular localization of the labelling was unclear): the cerebral cortex, the pyramidal cell layer of Ammon's horn, the granular cell layer of the dentate gyms, and the olfactory bulb. The present study revealed that in addition to these areas, cells containing a4 subunit mRNA were actually widely distributed throughout the entire brain.

Comparison of the localization of cells containing GABA~ receptor a~ and a~ subunit mRNA with those containing mRNA ]~r other subunits We have previously reported the detailed cellular localization of al 6,/~1_33° and ~,~ subunit mRNA expression in the brain. In addition, the localization of a2 subunit mRNA was also reported by Wisden et ai. ~s, although their results were not comprehensive. These reports taken together indicated that GABA^ receptor subunit mRNAs shows region-specific expression in the brain. For example, the cerebral cortex, pyramidal ceils of Ammon's horn, and granular cells of the dentate gyms express all the subunit mRNAs mentioned above. Purkinje cells in the cerebellar cortex express the al_2 ~,/]1,23° and y22 subunit mRHAs but lack a3-.4 and/]330 subunit mRNAs. The ventral thalamic nuclei show the strong expression of a~29 and j0230 subunit m~RNAs and the weak expression of a4,/]1 and/]23° subunit mRNAs but lack

neurons containing the /]330 and ~,22 subunit mRNAs. The medial thalamic nuclei contain cells expressing the a129, a3, a4 and/]2 subunit3° mRNAs at a moderate to strong intensity, while expression of the/]1, /]330 and y~ subunit mRNAs is relatively low. In the caudate-putamen, expression of a4 and /]330 mRNA is moderate to strong intensity, while expression of a3,/]1, /]23° and ~22 mRNA is very weak or absent. On the other hand, the globus pallidus expresses the a~29 and /]23o subunit mRHAs with a strong to moderate intensity, whereas expression of the a3,/]1,/]33o and y22 subunit mRNAs is weak or absent. The reticular thalamic nucleus express the ~,~ subunit with a high intensity and also express a3 mRNA but lack other subunit mRNAs. Table I summarizes localization of the different subunit mRNAs.

Roles of GABAA receptor subunits The differential localization of GABAA-receptor subunit mRNAs suggests that different subtypes of this receptor exist in different regions of the central nervous system. In order to elucidate the function of GABA, the roles of the various receptor subunits should be explored. For this puff ~se, the development of specific ligands for each subunit is necessary. At present, the functions of these subunits are quite obscure. All the subunits expressed in Xenopus oocytes show some kind of response to GABA 24. In addition, we have recently shown that as, a4 (Araki et al., in preparation) and f13~ subunit mRNAs are expressed at birth in many brain regions. At birth, the brain is still structurally and functionally immature, and neurogenesis is continuing in certain regions. There is evidence that GABA acts as both a trophic factor and a neurotransmitter during neurogenesis n~l. For example, GABAergic fibers and neurons are already seen in the fetal brain t'21, at a time when GABAergic synapses have not yet been established. In addition, it has been shown that GABA has atrophic effect on cultured cerebellar granule cells, and that this effect can be blocked by adding bicucculine methobromide (a GABAA-receptor antagonist) to the culture medium, suggesting that the trophic effect of GABA is mediated by the GABA^ receptor 13. Thus, the high level of expression of a3, a4, and/]3 subunit mRNAs in the neonatal rat brain may indicate that this subunit mediates the trophic effect of GABA during early brain development. We have recently demonstrated that many sites having cells which contain Y2subunit mRNA coincide with those containing GABA itself2, and we have also found that some GABAergic neurons contain ~'2 subunit mRNA in the thalamic reticular nucleus (Araki et al., in preparation). These findings saggest that the ~2 subunit is in-

312

volved in the GABA-GABA connections or that it is a component of the autoreceptor. In conclusion, the function of the subunits of the

GABA A receptor are still quite obscure and further analysis on this problem is required.

ABBREVIATIONS

lnt IO IP IPI LA La Lat LC LD LDTg LH LL LP LPB LPO LRt LSD LSI LSV LVe M MCPO MD MdD MDL MdV Me Me5 Med MG Mi MiA MiTg Mo5 MoL MPA MPB MS MTu MVe Pa Par PaS PBG PC PCL PCRt Pe PeF PH Pir PMD PMn Pn PnO PO Po Pr5 Pr$ PT PVA

3 6

7 10 12 AcbC AcbSh ACo AD AHA AI-IP AM Amb AOD AOE AOL AOM AOV APir APT Arc ATg AV BIC BST CAI-4 Ce CG

Cg CL CI CLi CM CPu Cu DC DEn DG Dk DLG DM DMSp5 DR DTg EP EPI EW Fr G Gi GL GI GP Gu lib HDB IC ICj IF IGL IGr IMLF

occulomotor nucleus abducens nucleus facial nucleus dorsal motor nucleus of vagus hypoglussal nucleus accumbens nucleus, core part accumbeus nucleus, shell part anterior corti.-~ amygdaloid nucleus anterodorsal thalamic nucleus anterior hypothalamic nucleus anterior hypothalamic area, posterior part anteromedial thalamic nucleus ambiguus nucleus anterior olfactory nucleus, dorsal part anterior olfactory nucleus, external part anterior olfactory nucleus, lateral part anterior olfactory nucleus, medial part anterior olfactory nucleus, ventral part amygdalopiriform transitional area anterior pretectal nucleus arcuate nucleus anterior tegmental nucleus anteroventral thalamic nucleus nucleus of the brachium of the inferior colliculus bed nucleus of the stria terminalis fields CAI-4 of Ammon's horn central am~gdaloid nucleus central gray cingulate cortex centrolateral thalamic nucleus claustrum caudal linial raphe nucleus central medial thalamic nucleus caudate putamen cuneate nucleus dorsal cochlear nucleus dorsal cndopiriform nucleus dentate gyrus nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsomedial hypothalandc nucleus dorsomedial part of the spinal trigeminal nucleus dorsal raphe nucleus dorsal tegmental nucleus entopeduncular nucleus external plexiform layer of the olfactory bulb Edinger-Westphal nucleus frontal cortex gelatinosus thalamic nucleus gigantocellular reticular nucleus granular layer of the cerebellum granular layer of the olfactory bulb globus pallidus gustatory thalamic nucleus habenular complex nucleus of the horizontal limb of the diagonal band inferior colliculus island of Calleja inteffascicular nucleus intergeniculate leaf internal granular layer of the olfactory bulb interstitial nucleus of the medial longitudinal fasciculus

R

Re Rh

interposed cerebellar nucleus inferior olivary nucleus interpeduncular nucleus internal plexiform layer of the olfactory bulb lateroanterior hypothalamic nucleus lateral amygdaloid nucleus lateral cerebellar nucleus locus coeruleus laterodorsal thalamic nucleus laterodorsal tegmental nucleus lateral hypothalamic nucleus lateral lemniscus lateral posterior thalamic nucleus lateral parabrachial nucleus lateral preoptic area lateral reticular nucleus lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral septal nucleus, ventral part lateral vestibular nucleus mammillary body magnocellular preoptic nucleus mediodorsal thalamic nucleus medullary reticular field, dorsal part mediodorsal thalamic nucleus, lateral part medullary reticular field, ventral part medial amygdaloid nucleus mesencephalic trigeminal nucleus medial cerebellar nucleus medial geniculate body mitral cell layer of the olfactory bulb mitral cell layer of the accessory olfactory bulb microcellular tegmental nucleus trigeminal motor nucleus molecular layer of the cerebellar cortex medial preoptic area medial parabrachial nucleus medial septal nucleus medial tuberal nucleus medial vestibular nucleus paraventral hypothalamic nucleus parietal cortex parasubiculum parabigeminal nucleus paracentral thalamic nucleus l~rkinje cell layer of the cerebellum parvocellular reticular nucleus periventricular hypothalamic nucleus perifornical nucleus posterior hypothalamic area piriform cortex dorsal part of the premammillary nucleus paramedian reticular nucleus pontine nucleus pontine nucleus, oral part preolivary nuclei posterior thalamic nuclear group principal trigeminal nucleus presubuculum paratenial thalamic nucleus paraventral thalamic nucleus, anterior part red nucleus reuniens thalamic nucleus rhomboid thalamic nucleus

313 RS Rt RtTg S SC SCH SI SNC SNR SO Sol SolC SP5C SPSI SP50

SpVe STh SubB

retrosplenial cortex reticular thaiamic nucleus reticulotegmehta! nucleus of the puns subicuinm superior coiliculus suprachiasmatic nucleus substantia innominata substantia nigra, pars compacta substantia nigra, pars reticulata supraoptic nucleus nucleus of the solitary tract comissural part of the nucleus solitary tract spinal ttigeminal nucleus, pars candalis spinal trigeminai nu¢|¢us, pars interpusitus spinal trigeminal nucleus, pars oralis spinal vestibular nucleus subthalamie nuciee~ subbrachial nucleus

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Subl SuM SuVe TI" Tu TZ VC VDB VM VMH VL VLG VP VPL VTA VTg Xi ZI

subincertal nucleus supramammillary nucleus superior vestibular nucleus tenia tecta olfactory tubercuinm trapezoid body ventral cochlear nucleus nucleus of the vertical limb of the diagonal band ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventrolateral thaiamic nucleus ventral geniculate nucleus ventral pallidum ventral posterolateral thalamic nucleus ventral tegrnental area ventral tegraental nucleus xipboid thalamic nucleus zona incerta

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