Expression of α2 adrenoceptors during rat brain development—I. α2A messenger RNA expression

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Neuroscience Vol. 76, No. 1, pp. 241–260, 1997 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00368-5

EXPRESSION OF á2 ADRENOCEPTORS DURING RAT BRAIN DEVELOPMENT—I. á2A MESSENGER RNA EXPRESSION U. H. WINZER-SERHAN,* H. K. RAYMON,† R. S. BROIDE,* Y. CHEN* and F. M. LESLIE*‡ *Department of Pharmacology, College of Medicine, University of California, Irvine, CA 92717, U.S.A. †Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037, U.S.A. Abstract––The distribution of á2A adrenoceptor messenger RNA expression in developing rat brain was characterized using in situ hybridization with 35S-labeled riboprobes. Intense hybridization signal was detected as early as embryonic day 14 in several areas adjacent to the forebrain and hindbrain germinal zones and in central noradrenergic neurons. A marked increase in messenger RNA expression was observed throughout the brain during late prenatal development, consistent with the migration and maturation of neurons in developing brain structures. In embryonic brain, there was a temporal and spatial correspondence in the appearance of á2A messenger RNA expression and binding sites labeled with [3H]idazoxan or p-[125I]iodoclonidine, indicating translation into receptor protein at an early stage of development. Whereas the presynaptic expression remained constant throughout development, there was an early postnatal decline of á2A receptor expression in many brain regions, including the olfactory bulb, cortex, caudate–putamen, hippocampus, thalamus, hypothalamus and medulla. Thereafter, messenger RNA expression increased, establishing an adult-like pattern during the second postnatal week, but remained low in areas such as the caudate–putamen, thalamus and hippocampus, which do not exhibit extensive expression in the adult. The transient perinatal expression of this á2 adrenoceptor type, which coincides with a period of hyperreactivity to sensory stimuli in the locus coeruleus, may indicate a specific functional role for the á2A receptor in the developing rat brain. The early and intense expression in olfactory structures suggests an involvement in early olfactory learning. The pattern of widespread, transient expression of á2A receptors in the fetal brain is in marked contrast to the postnatal development of the á2C receptor type. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: adrenergic receptor, noradrenaline, cortex, in situ hybridization, receptor autoradiography, olfactory bulb.

Noradrenergic innervation from the nucleus locus coeruleus (LC) occurs very early in the development of mammalian brain.33 In the rat, noradrenergic neurons differentiate at or before embryonic day 12 (E12) and give rise to projections shortly thereafter,62 reaching their destination prior to the differentiation of target neurons.57 Because of the early presence of norepinephrine (NE) in the developing brain, it has been suggested that the adrenergic system regulates several aspects of pre- and postnatal brain development, including cell division,60 neuronal maturation31,55,61 synaptogenesis5 and physiological plasticity.26 The physiological and biochemical responses of NE are mediated via three classes of receptor: á1, á2 and â. Of these, the â receptor has been examined most closely for its possible role in developmental

events.17,61 Fewer studies have investigated the possible roles of á1 or á2 receptors during brain ontogeny. In this and the subsequent paper72 we have examined the anatomical localization of á2 adrenoceptor mRNA and protein in rat brain during prenatal and postnatal development. This detailed analysis should provide a framework for subsequent functional studies on the role of á2 receptors in the developing brain.

‡To whom correspondence should be addressed. Abbreviations: E, embryonic day; EDTA, ethylenediaminetetra-acetate; LC, locus coeruleus; NE, norepinephrine; P, postnatal day. 241

á2 receptor pharmacology and localization Within the last decade, the existence of multiple á2 adrenoceptor subtypes has been confirmed both pharmacologically6,9 and through molecular cloning in human28,38,53,70 and in the rat.12,30,66,74 Although early studies suggested that there were at least four receptor subtypes,10 subsequent data have provided evidence for three unique receptor types encoded by separate genes (reviewed by Bylund et al.11). All three á2 adrenoceptors exhibit similar affinities for the endogenous ligands NE and epinephrine,22 and are

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negatively coupled to adenylyl cyclase.19,24 However, recent studies with cloned expressed receptors have indicated that the subtypes differ in their characteristics of ligand-induced desensitization.18,19 The distribution of á2 adrenoceptors in the adult rat brain has been investigated using receptor autoradiography,7,8,25,27,68 in situ hybridization41,45,56 and, to a more limited extent, immunohistochemical techniques.3,54 Recent autoradiographic studies have described the postnatal development of á2 adrenoceptors in the tree shrew21 and in developing Rhesus monkey neocortex.34 However, knowledge about á2 adrenoceptor distribution during rat brain development is sparse (reviewed by Duman and Alvaro17), even though this species represents an important model for developmental studies.15,20,23 Furthermore, those studies which have examined the ontogeny of á2 adrenoceptor protein or mRNA in rat brain have used gross dissection techniques, which do not provide much anatomical resolution.17,40,46,47 In this study we have used in situ hybridization and radioligand binding autoradiography to examine the regional expression of á2 adrenoceptor mRNA and protein in developing rat brain.

á2A receptors The á2A adrenoceptor was first characterized pharmacologically in binding studies of human platelets as a site with low affinity for prazosin and high affinity for oxymetazoline and rauwolscine.13 The gene encoding this receptor type was subsequently cloned from a human platelet library and designated á2C10, based on its location on human chromosome C10.28 A homologous gene was cloned from rat brain and named RG20.30 However, the pharmacology of the cloned expressed rat receptor was distinct from that of the human á2A receptor in that it had significantly lower affinity for the antagonist, rauwolscine. These findings are consistent with previous pharmacological identification of a site in rat tissues which was characterized either as a rauwoscine-insensitive adrenoceptor6 or, more commonly, as an á2D adrenoceptor.59 Recent molecular data have indicated, however, that the low affinity of the rat RG20 clone for yohimbine and rauwolscine is probably the result of a single amino acid exchange at position 20137 and that the protein encoded by this gene has a high degree of structural homology with the human á2A receptor encoded by á2C10.29 Thus, there is increasing evidence that the differing pharmacology of the rat and human proteins reflects species variance rather than the existence of distinct receptor subtypes.11 In the present study we have used cRNA probes transcribed from RG20 to localize á2A receptor mRNA within developing rat brain. In order to determine whether there is active translation from mRNA to protein in the embryonic brain, we have

compared the localization of á2A receptor mRNA with that of binding sites labeled by (3H)idazoxan. EXPERIMENTAL PROCEDURES

Materials The following materials were obtained from the sources indicated: bovine serum albumin, polyvinyl pyrrolidone, poly--lysine, RNase A and Cresyl Violet acetate (Sigma, St Louis, MO, U.S.A.); pBluescript II SK+ (Stratagene, La Jolla, CA, U.S.A.); Quick spin columns, proteinase K and yeast tRNA (Boehringer Mannheim Biochemicals, Indianapolis, IN, U.S.A.); formamide (Fluka, Ronkonkoma, NY, U.S.A.); dextran sulfate (Pharmacia, Piscataway, NJ, U.S.A.); sodium acetate (Fisher, Pittsburgh, PA, U.S.A.); Hyperfilm 3H and Hyperfilm â-max (Amersham, Arlington Heights, IL, U.S.A.); nuclear track emulsion (NTB-2), XAR film and D-19 developer (Kodak, Rochester, NY, U.S.A.); [35S]UTP and p-[125I]iodoclonidine (2200 Ci/mmol; Dupont NEN, Boston, MA, U.S.A.); [3H]idazoxan (41– 60 Ci/mmol; Amersham, Chicago, IL, U.S.A.), T7 and Sp6 RNA polymerases, RNAsin ribonuclease inhibitor, DNase, nucleotides and dithiothreitol (Promega Corp., Madison, WI, U.S.A.). Tissue preparation Timed pregnant Sprague–Dawley rat dams (Harlan, San Diego, CA, U.S.A.) were killed by decapitation and their embryos removed by Cesarean section on E14, E15, E17, E19 and E21. The heads of the embryos were frozen in isopentane at "20)C. Pups, at postnatal day 1 (P1), P3, P5, P7, P11, P14, P21 and adult rats were killed by decapitation and their brains quickly removed and frozen in isopentane at "20)C. For each age, at least two brains were used and sectioned in the transverse plane from anterior to posterior, and from posterior to anterior. Brains were stored at "80)C before use. For receptor binding, 20-µm tissue sections were cryostat cut and mounted on to ice-cold gelatin-coated slides. Sections were then dehydrated at 4)C for 2 h and stored desiccated at "20)C. Sections for in situ hybridization were mounted on to slides with an additional coating of poly-lysine, kept at "20)C, and postfixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4), for 1 h at 22)C. Sections were then washed in phosphate-buffered saline, air dried and stored desiccated at "20)C until use. In order to reduce inter-experimental variability, brains of different developmental ages were stored at "20)C for up to four weeks, then processed simultaneously for in situ hybridization or radioligand binding. Probe preparation A 3.0-kb genomic DNA fragment in pGEM7Zf(+), containing a 1380-bp sequence encoding the rat á2A (RG20) receptor, was kindly provided by Dr Kevin Lynch of the University of Virginia. [35S]UTP was used to synthesize cRNA probes from this template in both the sense and antisense orientations using T7 and SP6 RNA polymerases, respectively. Riboprobes were further subjected to alkaline hydrolysis to yield products with an average size of 600 bases.14 To test the specificity of the full-length probe, a 575-bp fragment from RG20 (778–1353 bp) was subcloned into pBluescript II SK+ using the Kpn I and Eco47 III restriction sites in RG20. This fragment, á2A(i3,4), encompassed a region within RG20 of low homology with other á2 adrenoceptors, extending from halfway into the third intracellular loop through the fourth transmembrane domain. In situ hybridization Tissue sections were processed for in situ hybridization according to a modification of the method described by

á2A adrenoceptor expression during rat brain development Simmons et al.58 Briefly, sections were pretreated with Proteinase K (0.1 mg/ml) for 10 min at 22)C, acetylated, dehydrated through graded ethanols and then air dried. Slide-mounted sections were incubated for 18 h at 60)C with a hybridization solution (50% formamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 500 mg/ml tRNA, 10 mM dithiothreitol, 0.3 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) containing riboprobes (1#107 c.p.m./ml) in either the antisense or sense orientation. Sections were incubated with RNase A (20 µg/ml) for 30 min at 37)C, followed by four 5-min high-stringency washes of decreasing salinity (0.5– 2#standard saline citrate buffer) at 22)C and a 30-min wash in 0.1#standard saline citrate at 65)C. Tissue sections were dehydrated and apposed to â-max film for three or 10 days for the full-length and á2A(i3,4) probes, respectively. Following film development, sections were dipped in liquid NTB emulsion for higher resolution anatomical analysis of mRNA localization. After an appropriate exposure period, slides were developed in Kodak D-19, fixed, counterstained with Cresyl Violet, coverslipped and analysed using transillumination microscopy. Additional adjacent sections of postnatal rat brains were stained with Cresyl Violet for higher anatomical resolution. Radioligand binding The binding conditions for [3H]idazoxan were similar to those described previously for rat brain tissue.6,52 Briefly, tissue sections were preincubated for 15 min at 22)C in 150 mM Na2KHPO4 buffer (pH 7.7), then incubated for 2 h at 4)C in buffer containing 0.8 nM [3H]idazoxan. Following incubation, sections were buffer washed twice for 1 min each time at 4)C. p-[125I]Iodoclonidine binding conditions were similar to those described by Wallace et al.67 Tissue sections were preincubated in Tris buffer (170 mM; pH 7.6) containing 20 mM MgCl2 for 20 min at 22)C, then incubated for a further 90 min at 22)C in the same buffer containing 0.2 nM p-[125I]iodoclonidine. For both [3H]idazoxan and p-[125I]iodoclonidine, non-specific binding was determined in alternate sections in the presence of phentolamine (1 µM). After washing, sections were dried thoroughly and desiccated for two days before apposition to tritium-sensitive film. After an appropriate exposure time, films were developed and tissue sections stained with Cresyl Violet for anatomical analysis. Data analysis The distribution of á2A adrenoceptor mRNA was evaluated in both films and emulsion-dipped sections, and that of [3H]idazoxan and p-[125I]iodoclonidine binding sites from autoradiographic films. Anatomical structures were identified by light-field analysis of Cresyl Violet-stained sections and compared to adult and prenatal rat brain atlases.2,48,49 The relative intensity of mRNA labeling was expressed as light (+), moderate (++) and high (+++) after thorough evaluation of both film and dark-field autoradiographic images. The relative intensities of á2 binding sites, labeled with [3H]idazoxan or p-[125I]iodoclonidine, in the forebrain during late prenatal and early postnatal ages were evaluated and expressed in a similar manner. RESULTS

Probe specificity and adult distribution The specificity of the full-length hydrolysed á2A probe was analysed by in situ hybridization in postnatal and adult rat brains. Identical patterns of mRNA distribution were found for the full-length á2A and the short á2A(i3,4) riboprobes in brain sections from adult, as well as from pups aged P11 and

243

P1 (data not shown). Sections incubated with riboprobes synthesized in the sense orientation exhibited only background levels of hybridization. Since the expression patterns were the same, the more sensitive full-length probe was chosen for this study. In adult rat brain, the pattern of á2A receptor mRNA expression which we observed was very similar to that which has been reported by others (Table 1B).41,45,56 The strongest hybridization signal was detected in the anterior olfactory nucleus (Fig. 1), the posterior amygdala (Fig. 1F), the pontine nucleus (Fig. 1G), the LC (Fig. 1H), the nucleus of the solitary tract, the dorsal motor nucleus of the vagus (Fig. 1I) and the lateral reticular nucleus (Fig. 1J). Moderately strong hybridization was found in all areas of the allo- and isocortex, piriform cortex, lateral septum (Fig. 1C), amygdala, hippocampus and hypothalamus, including the medial preoptic nucleus (Fig. 1D), interpeduncular nucleus (Fig. 1F) and several hindbrain nuclei (Fig. 1H–J). In the cerebellum, á2A receptor mRNA expression was limited to a few scattered cells in the granule cell layer (Fig. 1H). Low levels of á2A receptor mRNA expression were seen in the glomerular and internal granule layers of the olfactory bulb (Fig. 1A). Little hybridization was detected in the basal ganglia, dorsomedial hippocampus, thalamus and midbrain, except for the pontine nucleus and interpeduncular nuclei. á2A mRNA expression in fetal brain at mid-gestation At E14, the earliest time-point examined, a robust signal for á2A receptor mRNA was observed in areas adjacent to the forebrain, midbrain and hindbrain germinal zones, with the germinal zones exhibiting only light expression (Fig. 2). In the forebrain, intense á2A mRNA hybridization was detected in the striatal anlage, endopiriform nucleus, piriform cortex and in the pial cell layer, superficial to the cortical anlage (Fig. 2A). High levels of mRNA expression were also apparent in the hypothalamic anlage, pons, medulla and spinal cord, with moderate expression in the thalamus, mesencephalon and cerebellar anlage (Fig. 2B). á2A mRNA expression in fetal brain at late gestation During late embryonic development (E19 and E21), á2A mRNA expression was very intense in most structures of the developing rat brain, especially in the cortex, caudate–putamen, posterior mesencephalon, pons and medulla oblongata (Table 1, Fig. 3). The strongest signal was detected in the isocortex, LC and inferior olive. However, hybridization in the germinal zones remained very limited. Telencephalon In the olfactory bulb, á2A receptor mRNA was first detected with strong expression intensity in the accessory olfactory bulb and main olfactory bulb at

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Fig. 1. á2A adrenoceptor mRNA expression in adult rat brain. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (J). Scale bar=1000 µm. Abbreviations: 10, dorsal motor nucleus of the vagus; AON, anterior olfactory nucleus; BL, basolateral amygdaloid nucleus; BST, bed nucleus of the stria terminalis; CA1–3, hippocampal fields CA1–CA3 of Ammon’s horn; Cb, cerebellum; Ce, central amygdaloid nucleus; Cg, cingulate cortex; CPu, caudate–putamen; DM, dorsomedial hypothalamic nucleus; Ent, entorhinal cortex; Fr, frontal cortex; Gl, glomerular layer of the olfactory bulb; IC, frontal cortex; IGr, internal granule layer of the olfactory bulb; IP, interpeduncular nucleus; LC, locus coeruleus; LH, lateral hypothalamic area; LO, lateral orbital cortex; LRt, lateral reticular nucleus; LS, lateral septum; Me, medial amygdaloid nucleus; Mo5, motor trigeminal nucleus; MPO, medial preoptic nucleus; Oc, occipital cortex; Par, parietal cortex; PCo, posteriomedial cortical amygdaloid nucleus; Pir, piriform cortex; PRh, perirhinal cortex; PV, paraventricular thalamic nucleus; RS, retrosplenial cortex; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; Te, temporal cortex.

á2A adrenoceptor expression during rat brain development

245

Table 1. The distribution of á2A mRNA during rat brain development Brain area

E19

E21

P1

P3

P5

P7

P11

P14

P21

Adult

Olfactory system Olfactory bulb (OB) Anterior olfactory n (AON) Piriform cortex (Pir) Tenia tecta (TT) Claustrum (Cl) Endopiriform n (En)

na. + +++ +++ ++ +++

++ +++ +++ ++ ++ +++

+ +++ ++ ++ + +

++ +++ +++ ++ +++ ++

++ +++ +++ ++ +++ ++

++ +++ +++ ++ +++ ++

+++ +++ +++ +++ +++ ++

+++ +++ +++ +++ +++ ++

+++ +++ +++ +++ ++ ++

+ +++ ++ +++ ++ ++

Allocortex Infralimbic med front (IL) Cingulate (Cg) Retrosplenial (RSA/G) Insular (ICx) Perirhinal (PRh)

+ +++ +++ + +

+ ++ ++ + +

+ + + + +

+ + + + +

++ + ++ ++ ++

++ ++ ++ ++ ++

++ +++ +++ ++ ++

++ +++ +++ ++ ++

++ ++ ++ ++ ++

+ ++ ++ ++ +

Isocortex Frontal (Fr) Parietal (Par) Temporal (Te) Occipital (Oc)

++ +++ +++ ++

++ +++ +++ ++

++ +++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

++ ++ ++ ++

Basal ganglia Caudate–putamen (CPu) Ventral pallidum (VP) Fundus striati (FStr) Nucleus accumbens (Acb)

+++ + + ++

++ ++ ++ ++

+ + + +

+ + + +

+ + + +

+ + + +

+ + + +

+ + + +

— — — —

— — — —

Septum Lateral septum (LS) Medial septum (MS) Septohippocampal n (SHi) Diagonal band (H/VDB)

+++ + +++ —

++ + +++ —

+++ + ++ +

+++ + ++ +

+++ + ++ +

+++ ++ ++ +

+++ ++ ++ +

+++ ++ ++ +

+++ ++ ++ +

+++ + ++ —

Hippocampus Dentate gyrus (DG) CA1 CA3 Subiculum (S) Presubiculum (PrS) Entorhinal cortex (Ent)

— +++ +++ ++ ++ +

— +++ +++ ++ ++ +

++ ++ ++ ++ + ++

++ ++ ++ ++ ++ ++

+ ++ ++ ++ +++ ++

+ ++ ++ ++ +++ +++

+ +++ +++ ++ +++ +++

+ +++ +++ ++ +++ +++

+ +++ +++ ++ +++ ++

+ ++ ++ + ++ ++

Amygdaloid complex Olfactory (AA, ACo, APir) Lat olfactory tract n (LOT) Medial amygdaloid n (Me) Post/Med cortical n (PMCo) Amyg/Hipp trans area (AHi) Basomedial nucleus (BM) Basolateral nucleus (BL) Central nucleus (Ce) Bed n stria terminalis (BST)

+++ ++ ++ + ++ — ++ +++ ++

++ +++ + ++ ++ + + ++ ++

+ +++ + + + + + ++ ++

+ +++ + + + + ++ ++ ++

+ +++ + ++ ++ + + ++ ++

++ +++ + ++ ++ + + ++ ++

++ + + ++ ++ + + ++ ++

++ + + ++ ++ + + ++ ++

+ + + ++ ++ + + ++ ++

+ — + ++ ++ + + + ++

Thalamus Anterodorsal nucleus (AD) Mediodorsal nucleus (MD) Paraventricular n (PV) Ventral basal complex (VB) Lateral dorsal (LD) Dorsolat geniculate (DLG) Ventrolat geniculate (VLG) Lateral posterior n (LP) Zonia incerta (Zl)

++ ++ +++ — ++ ++ ++ ++ +

++ + +++ — + ++ ++ ++ ++

+ + ++ — + + — + +

+ + ++ — + + — + +

+ + ++ + + + — + +

+ + ++ + — — + — +

— + ++ — — — ++ — +

— + ++ — — — ++ — +

— + + — — — + — +

— — + — — — + — +

Hypothalamus Medial preoptic n (MPO) Lateral preoptic area (LPO) Anterior hyp area (AH) Paraventricular hyp n (Pa) Ventromedial hyp (VMH) Dorsomedial hyp (DM) Arcuate nucleus (Arc)

+ + ++ +++ + +++ —

+ + ++ +++ + ++ —

++ + + ++ + ++ —

+ + + ++ + +++ —

++ + + +++ + +++ —

+++ ++ + +++ + +++ —

+++ ++ + + + +++ —

+++ ++ + + + +++ +

++ + + ++ + ++ +

++ + + +++ + ++ +

246

U. H. Winzer-Serhan et al. Table 1. Continued

Brain area

E19

E21

P1

P3

P5

P7

P11

P14

P21

Adult

Hypothalamus Supramammillary n (SuM) Medial mammilliary n (MM) Lateral hyp area (LH) Septohyp nucleus (SHy) Striohyp nucleus (STHy)

++ ++ ++ +++ +++

+ + ++ +++ +++

+ + ++ +++ ++

+ + ++ +++ ++

+ + ++ +++ ++

+ + +++ +++ +++

++ ++ +++ ++ ++

++ ++ +++ ++ +

++ + ++ ++ +

++ + ++ ++ +

Midbrain Superior colliculus (SC) Inferior colliculus (SC) Interpeduncular n (IP) Mesenceph trigem n (Me5) Dorsal raphe n (DR) Central gray (CG)+ Pontine nuclei (Pn)

+++ +++ + ++ ++ +++ +++

+++ +++ ++ ++ +++ +++ +++

++ + +++ + + + +++

+++ + +++ + + ++ +++

+++ ++ +++ + + ++ +++

+++ ++ +++ + + ++ +++

+++ ++ +++ + + ++ +++

++ ++ +++ + + ++ +++

+ + +++ + + + +++

+ + + + + + +++

Pons Lat parabrachial n (LPB) Locus coeruleus (LC) A5 area A4 area Motor trigeminal n (Mo5) Reticulotegmental n (RtTg) Preposit hypoglos n (PrH) Parvocell reticular n (PCRt) Trigeminal nucleus (Pr5) Ventral cochlear n (VC)

+++ +++ ++ ++ ++ ++ ++ ++ ++ ++

+++ +++ +++ ++ ++ ++ ++ ++ ++ ++

++ +++ +++ ++ + + + + + ++

++ +++ +++ ++ + + ++ + + +

++ +++ +++ ++ + + ++ + ++ ++

+++ +++ +++ ++ + + +++ ++ ++ ++

+++ +++ +++ ++ ++ + +++ ++ ++ ++

++ +++ +++ ++ ++ ++ +++ ++ ++ ++

+ +++ +++ ++ + + ++ + ++ +

+ +++ +++ ++ + + ++ + ++ +

Brain stem Nucleus solitary tract (Sol) Hypoglossal nucleus (12) Dorsal motor n. vagus (10) External cuneate n. (ECu) Int reticular nucleus (IRt) Inferior olive (IO) Lat paragigantocell (LPGi) Medullary recicular n (MdD) Lateral reticular n. (LRt) Spinal trigeminal n. (Sp5)

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

++ + + ++ + +++ + ++ + ++

++ ++ + ++ + +++ + ++ + ++

+++ ++ + ++ + +++ + ++ + ++

+++ ++ +++ +++ ++ +++ ++ ++ ++ +++

+++ + +++ +++ ++ ++ +++ ++ +++ +++

+++ + +++ +++ ++ + +++ ++ +++ +++

+++ + +++ ++ + — +++ ++ +++ ++

+++ + +++ ++ ++ — ++ + +++ ++

Cerebellum Lateral cerebellar n. (Lat) Internal granule layer

++ n.d.

++ n.d.

+ n.d.

+ n.d.

+ —

++ +

++ ++

+ ++

+ ++

— +

Spinal Cord Dorsal horn (DH) Ventral horn (VH)

+++ +++

+++ +++

++ +

++ +

+++ +

+++ ++

+++ ++

+++ +

++ +

n.d. n.d.

Regional distribution of á2A adrenoceptor mRNA expression during rat brain development, from E19 to adult. The relative density of á2A mRNA expression is indicated: (—) no signal detected; (+) low; (++) moderate; (+++) high; n.d. not determined.

E21 (Table 1). In the neocortex, especially high levels of mRNA expression were detected in the parietal and temporal cortices, starting at E19 (Fig. 3B, D). The increased expression was mainly found in the subventricular and intermediate zones, as well as in the upper part of the cortical plate. The insular cortex ventral to the parietal cortex exhibited low signal, whereas mRNA expression in the cingulate cortex was increased during late prenatal development (Fig. 3A, B). The hippocampus exhibited high transcript levels in the dorsomedial and lateral parts of the CA1 and in the subiculum (Fig. 3C, D), with expression also detected in CA3 by E21 (Table 1). In

the amygdala, expression was higher in anterior than posterior nuclei, with especially striking hybridization signal in the nucleus of the lateral olfactory tract. In contrast, mRNA expression in the basal ganglia decreased during late gestation, with a rostral to caudal gradient (Fig. 3A, B). Diencephalon Increased mRNA expression was observed at E17 in both the thalamus and hypothalamus. At E19, á2A receptor mRNA was found at moderately high levels in several thalamic nuclei (Table 1). In the hypothalamus, expression was intense and widespread,

á2A adrenoceptor expression during rat brain development

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Fig. 2. á2A adrenoceptor mRNA expression in developing brain at E14. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at the level of the forebrain (A) and hindbrain (B). Scale bar=100 µm. Abbreviations: Cb, cerebellum; CPu, caudate–putamen; Hy, hypothalamus; Md, medulla; ne, neuroepithelium; Pir, piriform cortex; Po, pons; SpC, spinal cord; Th, thalamus.

especially in the paraventricular, dorsomedial and striohypothalamic nuclei. The ventromedial hypothalamic nucleus and arcuate nucleus showed only weak hybridization. However, the arcuate neuroepithelium expressed á2A receptor mRNA starting during late embryonic development.

Mesencephalon By E19, á2A receptor mRNA was widespread throughout the midbrain, with a caudal to rostral gradient. The strongest hybridization signal was detected in the caudal superior colliculus, central gray, dorsal raphe nucleus, interpeduncular nucleus, inferior colliculus and pontine nuclei (Fig. 3E–G, Table 1).

Rhombencephalon Strong á2A receptor mRNA expression was detected in the pons and brainstem throughout prenatal development. However, there was a marked increase in expression by E17, with very intense signal by E19 (Table 1). Especially robust hybridization was found in the LC, hypoglossal nucleus, dorsal cochlear nucleus, inferior olive and spinal cord (Fig. 3E–G). During late prenatal development, á2A mRNA expression in the cerebellum was limited to the lateral cerebellar nuclei (Fig. 3G).

Prenatal á2 adrenoceptor binding sites Specific [3H]idazoxan and p-[125I]iodoclonidine binding was detected as early as E15, increasing throughout prenatal development. The binding patterns of the two ligands were practically identical in their spatial and temporal distributions (Table 2). By E17, very prominent receptor labeling was found in the piriform cortex and caudate–putamen (Fig. 4A). Moderate to light levels of binding were also detected in the hippocampus, thalamus, hypothalamus and pons (Fig. 4B, C). At E21, [3H]idazoxan receptor labeling was clearly visible in the olfactory bulb (Fig. 4D). Cortical labeling remained moderate, except for the parietal and temporal cortices, where high levels of binding were detected in the deeper layers above the germinal zone (Fig. 4F). Labeled sites were also detected along the midline of the forebrain, which most likely corresponded to ascending noradrenergic fibers which pass through that area (Fig. 4E). mRNA expression in the early postnatal period By P1, á2A mRNA expression had declined in many structures (Table 1). Several areas that continued to exhibit moderate to high levels of expression, similar to their prenatal pattern, were the anterior olfactory nucleus (Fig. 5B), lateral septum (Fig. 5C), pontine nucleus (Fig. 5G), and several nuclei in the pons and brainstem (Fig. 5H–J). All of these

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Fig. 3. á2A adrenoceptor mRNA expression in developing brain at E19. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (G). Scale bar=1000 µm. Abbreviations: 12, hypoglossal nucleus; AA, anterior amygdala; BST, bed nucleus of the stria terminalis; CA1, hippocampal field CA1 of Ammon’s horn; CG, central gray; Cg, cingulate cortex; CPu, caudate–putamen; DC, dorsal cochlear nucleus; DM, dorsomedial hypothalamic nucleus; ICx, insular cortex; IC, inferior colliculus; IO, inferior olive; Lat, lateral cerebellar nucleus; LC, locus coeruleus; LD, lateral dorsal thalamic nucleus; LP, lateral posterior thalamic nucleus; LS, lateral septum, Md, medulla; Par, parietal cortex; Pir, piriform cortex; Pr5, principal sensory trigeminal nucleus; RS, retrosplenial cortex; S, subiclulum; SC, superior colliculus; SpC, spinal cord; Te, temporal cortex.

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Table 2. The distribution of á2 adrenoceptor radioligand binding sites in developing rat forebrain Brain area

E19

E21

P1

P3

P5

P7

Olfactory system Olfactory bulb Anterior olfactory nucleus Piriform cortex Tenia tecta Claustrum/endopiriform nucleus

n.d. + ++ + ++

++ ++ ++ ++ +++

++ ++ ++ ++ +

+ ++ ++ ++ ++

+ ++ ++ ++ ++

+ ++ ++ ++ ++

Isocortex Parietal Temporal

++ ++

+++ +++

++ ++

++ ++

+ +

+ +

Basal ganglia Anterior commissure Nucleus accumbens Fundus striati Caudate–putamen

" " + ++

+ " + ++

++ " + +

+++ + + +

+++ + + +

++ + + +

Septum Medial septum Lateral septum Septohippocampal nucleus

" + +

" ++ ++

" ++ ++

+ ++ +++

+ ++ +++

+ ++ +++

Hippocampus CA1

+

+++

++

+

+

+

The distribution of á2 adrenoceptor binding sites in selected forebrain regions during late prenatal and early postnatal development is given. The relative density of á2 binding sites is expressed as indicated: ", no signal detected; +, low; ++, moderate; +++, high; n.d., not determined.

brain structures, except for the inferior olive, had high levels of expression throughout postnatal development and in the adult (Table 1). Telencephalon In the cortex, á2A receptor mRNA expression was detected in all regions, with the lowest levels found in the cingulate, retrosplenial and insular cortices (Fig. 5B, C, E), moderate levels in the occipital and temporal cortices (Fig. 5E), and the highest levels in the parietal cortex (Fig. 5D). However, expression was lower than that observed prenatally. By P1, there was strong mRNA expression in the superficial part of the cortical plate, especially in the parietal cortex, as well as throughout the subplate. In the hippocampus, hybridization in the dorsomedial and ventral hippocampal CA1 and CA3 regions was moderately high at P1, with light signal now detectable in the dentate gyrus (Fig. 5D, E). In the amygdala, á2A receptor mRNA expression was widespread, even though it had declined to low levels in most nuclei. However, expression remained high in the central amygdaloid nucleus, bed nucleus of the stria terminalis and nucleus of the lateral olfactory tract (Fig. 5D, E). Within the basal ganglia, á2A mRNA had declined to very low levels, except for the fundus striati and nucleus accumbens (Fig. 5B, C). Diencephalon The expression in the diencephalon had greatly declined by P1. In the thalamus, only the para-

ventricular thalamic nucleus and medial thalamic nuclei exhibited low levels of á2A mRNA. Expression in the hypothalamus was more widespread and remained moderately high in several nuclei, with the highest expression in the striohypothalamic nucleus (Fig. 5D, E). Mesencephalon In the midbrain, á2A mRNA expression had greatly declined by P1, but many scattered cells throughout the midbrain still exhibited intense hybridization. Only the pontine nucleus, interpeduncular nucleus and the superficial layer of the superior colliculus exhibited high levels of expression, whereas the signal had greatly declined in the dorsal raphe nucleus, central gray and inferior colliculus (Fig. 5F, G). Rhombencephalon By P1, hybridization in the pons and brainstem had greatly declined in many nuclei, but remained widespread, due to scattered cells with strong expression (Table 1). The most intense expression was detected in the LC and inferior olive, with moderately high expression in the external cuneate nucleus, ventral cochlear, nucleus of the solitary tract and hypoglossal nucleus, as found prenatally. In the spinal cord, á2A receptor mRNA expression had strongly decreased in the ventral horn, but remained moderately high in the dorsal horn (Fig. 5K). In the cerebellum, expression was restricted to the lateral cerebellar nucleus (Fig. 5H).

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Fig. 4. á2 adrenoceptor binding site distribution in embryonic rat brain labeled with [3H]idazoxan. Computer-generated autoradiographic images of 20-µm-thick coronal sections at E17 (A–C) and E21 (D–G) are shown. Scale bar=1000 µm. Abbreviations: Co, cornea; CPu, caudate–putamen; Cx, cortex; FT, fiber tract; Hi, hippocampus; Hy, hypothalamus; NCa, nasal cavaty; OB, olfactory bulb; Par, parietal cortex; Pir, piriform cortex; Po, pons; Th, thalamus.

First postnatal week An increase in á2A receptor mRNA expression was found in many brain structures by the end of the first postnatal week. The highest hybridization intensity was seen in the anterior olfactory nucleus, piriform cortex, presubiculum, pontine nucleus, LC and inferior olive. However, very intense expression was also evident in many other structures throughout the

brain, except for the basal ganglia and thalamus, where only low hybridization signal was detected (Table 1). Telencephalon In the main olfactory bulb, two bands of hybridization signal were detected at P7, corresponding to the glomerular and internal granule cell layers (Fig. 6A).

á2A adrenoceptor expression during rat brain development

Fig. 5. á2A adrenoceptor mRNA expression in one-day-old rat brain. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (K). Scale bar=1000 µm. Abbreviations: A5, A5 noradrenaline area; AON, anterior olfactory nucleus; CA1–3, hippocampal fields CA1–CA3 of Ammon’s horn; Ce, central amygdaloid nucleus; CG, central gray; Cg, cingulate cortex; Cl, claustrum; DM, dorsomedial hypothalamic nucleus; DG, dentate gyrus; DH, dorsal horn; ECu, external cuneate nucleus; Ent, entorhinal cortex; Fr, frontal cortex; IC, inferior colliculus; IO, inferior olive; IP, interpeduncular nucleus; Lat, lateral cerebellar nucleus; LC, locus coeruleus; LOT, nucleus of the lateral olfactory tract; LS, lateral septum; MPO, medial preoptic nucleus; OB, olfactory bulb; Oc, occipital cortex; Par, parietal cortex; Pir, piriform cortex; PN, pontine nuclei; SC, superior colliculus; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; Te, temporal cortex; VC, ventral cochlear nucleus; VH, ventral horn.

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Fig. 6. á2A adrenoceptor mRNA expression in seven-day-old rat brain. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (K). Scale bar=1000 µm. Abbreviations: AI, agranular insular cortex; AON, anterior olfactory nucleus; CA3, hippocampal field CA3 of Ammon’s horn; Cb, cerebellum; Cg, cingulate cortex; CPu, caudate–putamen; DH, dorsal horn; ECu, external cuneate nucleus; En, endopiriform nucleus; Ent, entorhinal cortex; Fr, frontal cortex; FStr, fundus striati; IC, inferior colliculus; IO, inferior olive; IP, interpeduncular nucleus; LC, locus coeruleus; MPO, medial preoptic nucleus; OB, olfactory bulb; Oc, occipital cortex; Pa, paraventricular hypothalamic nucleus; Par, parietal cortex; Pir, piriform cortex; Pn, pontine nuclei; PRh, perirhinal cortex; PV, paraventricular thalamic nucleus; SC, superior colliculus; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; StHy, striohypothalamic nucleus; Te, temporal cortex; VH, ventral horn; VL, ventrolateral thalamic nucleus.

á2A adrenoceptor expression during rat brain development

Fig. 7. á2A adrenoceptor mRNA expression in 14-day-old rat brain. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (K). Scale bar=1000 µm. Abbreviations: 10, dorsal motor nucleus of the vagus; aca, anterior commissure, anterior part; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; BST, bed nucleus of the stria terminalis; CA1, 3, hippocampal fields CA1 and CA3 of Ammon’s horn; CG, central gray; Cg, cingulate cortex; DH, dorsal horn; En, endopiriform nucleus; Ent, entorhinal cortex; Fr, frontal cortex; IO, inferior olive; IP, interpeduncular nucleus; LC, locus coeruleus; LH, lateral hypothalamic area; LO, lateral orbital cortex; LRt, lateral reticular nucleus; LS, lateral septum; MD, mediodorsal thalamic nucleus; Me, medial amygdaliod nucleus; MPO, medial preoptic nucleus; OB, olfactory bulb; Oc, occipital cortex; Par, parietal cortex; PCo, posteriomedial cortical amygdaloid nucleus; Pir, piriform cortex; Pn, pontine nuclei; PrS, presubiculum; RS, retrosplenial cortex; RSA, retrosplenial agranular cortex; RtTg, reticulotegmental nucleus of the pons; SC, superior colliculus; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; Te, temporal cortex.

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Fig. 8. á2A adrenoceptor mRNA expression in 21-day-old rat brain. Computer-generated autoradiographic images of 20-µm-thick coronal sections are shown at different levels of the brain, from rostral (A) to caudal (K). Scale bar=1000 µm. Abbreviations: 10, dorsal motor nucleus of the vagus; AON, anterior olfactory nucleus; Cb, cerebellum; Ce, central amygdaloid nucleus; CG, central gray; Cg, cingulate cortex; DH, dorsal horn; Ent, entorhinal cortex; Fr, frontal cortex; IP, interpeduncular nucleus; LC, locus coeruleus; LH, lateral hypothalamic area; LO, lateral orbital cortex; LRt, lateral reticular nucleus; LS, lateral septum; Me, medial amygdaloid nucleus; MPO, medial preoptic nucleus; OB, olfactory bulb; Oc, occipital cortex; Par, parietal cortex; PCo, posteriomedial cortical amygdaloid nucleus; Pir, piriform cortex; Pn, pontine nuclei; RS, retrosplenial cortex; Sol, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus; Te, temporal cortex.

á2A adrenoceptor expression during rat brain development

In the cortex, á2A receptor mRNA expression was detected with moderately high intensity in the iso- and allocortex, as well as in the claustrum and endopiriform nucleus (Fig. 6C). Hybridization signal remained limited only in the granular cingulate and retrosplenial cortices (Fig. 6D, E). In the parietal cortex, the transient up-regulation of expression in the cortical plate had declined, but expression was still elevated in layers V and VI. Throughout the remaining isocortex, moderate mRNA expression was detected in all layers, with stronger expression in layer IV and the subplate. In the hippocampus, á2A receptor mRNA expression markedly decreased in the dorsomedial hippocampal CA1 region and the dentate gyrus after P1 and P3, respectively (Fig. 6E). However, hybridization signal remained moderately high in the CA3 and ventral hippocampal CA1 region (Fig. 6F). In the entorhinal cortex and presubiculum, a strong increase in expression was detected during the first postnatal week (Fig. 6G). Diencephalon Whereas á2A receptor mRNA expression remained very limited in the thalamus, several hypothalamic nuclei exhibited strong expression (Table 1). In the medial preoptic nucleus, mRNA expression declined immediately after birth, but increased again to moderately high levels by the end of the first postnatal week (Fig. 6D). Mesencephalon In the midbrain, the á2A mRNA expression pattern remained largely unchanged, with numerous scattered cells throughout the midbrain exhibiting high expression (Fig. 6F, G). In the inferior colliculus, hybridization signal had increased to moderate levels by P7 (Fig. 6H). Rhombencephalon By P7, á2A receptor mRNA could be detected in nearly all pons and brainstem nuclei, with moderate to high levels of expression. However, some nuclei exhibited especially strong hybridization, including the LC, A5 area and inferior olive (Fig. 6H, I). In the spinal cord, expression remained unchanged in the dorsal horn, but was somewhat increased in the ventral horn (Fig. 6J). In the cerebellum, expression increased to moderately high intensity in the lateral nucleus. At the same time, á2A mRNA could be detected in scattered cells in the granule layer (Fig. 6H). á2 adrenoceptor binding in the first postnatal week During the first postnatal week, á2 adrenoceptor binding remained constant or increased in intensity in areas such as the anterior olfactory nucleus, piriform

255

cortex and lateral septum, which continued to exhibit high levels of á2A receptor mRNA expression (Table 2). However, binding declined in other areas, including the olfactory bulb, parietal and temporal cortices, caudate–putamen and the hippocampal CA1. There was an appearance of receptor labeling in the fiber tract of the anterior commissure during this period. This was not observed in the adult (data not shown). mRNA expression during the second postnatal week mRNA expression remained the same in most areas of the forebrain, midbrain and hindbrain. The highest signal was detected in the anterior olfactory nucleus, piriform cortex, presubiculum, pontine nucleus and LC; however, several other nuclei also showed strong expression (Table 1). The lowest level of expression was in the basal ganglia, thalamus and dorsal hippocampus. The distribution pattern was identical to that at P7 in most parts of the brain. However, changes were detected in some areas (Table 1, Fig. 7). Telencephalon In the olfactory bulb, the pattern of expression remained unchanged, although signal intensity increased to moderately high levels during the second postnatal week (Fig. 7A). In the cortex, mRNA distribution was generally similar to that at P7, except for a significant increase in mRNA expression in the agranular cingulate and retrosplenial cortices (Fig. 7D, E). In the amygdala, the transient increased mRNA expression in the nucleus of the lateral olfactory tract decreased to low levels during the second postnatal week (Table 1). Expression in other nuclei remained high, however, particularly in the posterior amygdala (Fig. 7F). Diencephalon In the thalamus, mRNA expression remained the same as that at P7. In the hypothalamus, cells with moderate levels of expression were scattered throughout. Strong signal remained in the medial preoptic nucleus, dorsomedial hypothalamic nucleus and lateral hypothalamic area, whereas it declined in the septohypothalamic, paraventricular and striohypothalamic nuclei to moderate and low levels of expression, respectively (Fig. 7D, E). Mesencephalon á2A receptor mRNA expression remained constant, with moderate to high levels found in scattered cells throughout. The expression in the interpeduncular and pontine nuclei remained very strong, but declined to moderate levels in the superior colliculus (Fig. 7F, G).

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Rhombencephalon Whereas the expression pattern remained the same in most areas of the hindbrain, the overall intensity decreased due to fewer cells with intense mRNA expression. However, changes were detected in the hypoglossal nucleus and spinal trigeminal nucleus and, most dramatically, in the inferior olive, where á2A mRNA expression declined to low levels during the second postnatal week (Fig. 7I). In contrast, expression in the lateral reticular nucleus rose from low to high intensity during this time (Fig. 7J). In the spinal cord, hybridization remained at high levels in the dorsal horn, but decreased in the ventral horn (Fig. 7K). In the cerebellum, mRNA expression increased in scattered cells of the granule layer, but decreased in the lateral nucleus (Fig. 7H). mRNA expression during the third postnatal week In most brain areas á2A mRNA expression remained constant. However, a few changes took place during the third postnatal week. In the cortex, the transient increase in expression in the agranular cingulate and retrosplenial cortices declined (Fig. 8D, E). In the thalamus, the expression in the few nuclei where á2A mRNA was detected dropped to low levels (Fig. 8D). In the midbrain, an overall decline in á2A mRNA expression took place during the third postnatal week, including the central gray, superior colliculus and inferior colliculus, but a rostral to caudal gradient, with stronger signal in the caudal part, remained (Fig. 8F, G). Strong hybridization was still detected in the pontine and interpeduncular nuclei, the latter decreasing to low levels after P21 (Fig. 8G). Signal intensity decreased throughout the pons, brainstem and spinal cord, except for nuclei with high levels of expression (Fig. 8H–K). In the cerebellum, á2A receptor mRNA expression declined in the lateral nucleus, but remained constant in a few scattered cells in the granule layer (Fig. 8H). By P21, the á2A mRNA expression pattern reflected the adult distribution, although expression intensity was generally higher than in the adult, especially in the olfactory bulb, subiculum, entorhinal cortex and interpeduncular nucleus (Table 1). DISCUSSION

In the present study, we have demonstrated an early developmental appearance of á2A adrenoceptors in the rat brain. At E14, the earliest timepoint examined, extensive mRNA expression was detected throughout the brain. Expression increased until late gestation and then, in many regions, declined after birth. In some areas, such as the basal ganglia and thalamus, expression consistently declined throughout postnatal development to low adult levels, whereas in other areas there was a more complex developmental profile. Embryonic mRNA expression was accompanied by a concomitant ap-

pearance of [3H]idazoxan and p-[125I]iodoclonidine binding sites, indicating active protein translation. The transient expression of á2A adrenoceptors at a critical period of neuronal differentiation suggests a potentially important role for this receptor in modulating brain development.

Technical considerations We have used a full-length, hydrolysed cRNA probe to examine the developmental expression of á2A adrenoceptor mRNA by in situ hybridization. Non-specific hybridization, as determined in alternate sections with probe transcribed in the sense orientation, was very low. Probe specificity was further confirmed by the use of a short á2A(i3,4) probe, directed against a non-homologous sequence of the á2A receptor, which yielded a hybridization pattern identical to that of the long probe. The use of a long riboprobe in the present study has provided increased sensitivity in comparison to the oligonucleotide probes used in some previous studies of adult rats.41,45 However, the pattern of expression in the adult which we observe is very similar to that reported previously with oligonucleotide probes41,45 and almost identical to results obtained with the same riboprobe.56 In contrast to the study of Scheinin et al.,56 however, we have detected only specific hybridization in the olfactory bulb, hippocampus and cerebellum, areas which were previously reported to exhibit non-specific labeling. The early widespread expression of á2A adrenoceptor mRNA in differentiating regions of the developing brain is paralleled by an early appearance of á2 receptor binding sites detected with [3H]idazoxan and p-[125I]iodoclonidine. Binding was especially strong in areas of intense á2A mRNA expression, like the piriform cortex, caudate–putamen and hindbrain. [3H]Idazoxan and p-[125I]iodoclonidine have been shown in previous studies to label the á2A receptor, and other á2 receptor subtypes, with high affinity.1,16,22 Although there are data to indicate that [3H]idazoxan may also label a non-adrenergic idazoxan binding site,42 we and others have demonstrated that binding to this additional site is highly sensitive to incubation buffer conditions.52,67 With the assay conditions used in the present study, [3H]idazoxan selectively labels á2 adrenoceptors.6,52 The spatial and temporal correspondence which we have observed between á2 receptor binding and mRNA expression strongly suggests the early appearance of á2A adrenoceptor protein. This conclusion is supported by the finding that [3H]rauwolscine, which exhibits high affinity for á2B and á2C adrenoceptor subtypes but low affinity for rat á2A,23 exhibits very limited binding in embryonic brain (see accompanying paper). However, we have not yet carried out pharmacological studies to definitively support this conclusion.

á2A adrenoceptor expression during rat brain development

Presynaptic á2A autoreceptor Recent pharmacological studies have shown that most presynaptic á2 receptors in the adult rodent brain are of the á2A subtype.36,65 This finding is supported by immunohistochemical studies, using subtype-specific antibodies, which have clearly shown the presence of á2A receptor protein in the LC and other noradrenergic cells,3,54 and by in situ hybridization studies showing the expression of á2A, but not á2B or á2C, mRNA in noradrenergic nuclei.45,56 We now demonstrate that the LC and other noradrenergic cell groups express á2A mRNA during fetal development, as early as E14. This finding suggests that there is an early developmental onset of presynaptic autoreceptor control of NE release. Both neurotransmitter release51 and electrophysiological studies44 have confirmed the presence of functional á2 receptors on embryonic LC neurons. Our present data suggest that these autoreceptors are of the á2A type. Postsynaptic á2A mRNA expression In addition to the early appearance of á2A adrenoceptor mRNA in noradrenergic nuclei, there is widespread cellular expression of mRNA throughout the embryonic brain. This is accompanied by expression of [3H]idazoxan binding sites, indicating that there is protein translation. Unlike primate brain, in which á2A adrenoceptor expression has been noted in germinal zones of active cell division,33,35 expression in the rat brain is restricted to areas adjacent to these proliferative zones. In many regions, receptor expression follows a developmental time-course which largely corresponds to the onset of neuronal migration and differentiation. Thus, at E14 there is intense mRNA expression in the striatum, hypothalamus and brainstem, whereas expression in later developing structures, such as the cortex and hippocampus, exhibits a later onset. In general, there is a decline in the intensity of mRNA expression around birth, when the process of migration and initial differentiation is complete. This is then followed, in some areas, by an increase in mRNA expression which appears in the adult pattern. Although this is the first detailed regional study of á2A adrenoceptor mRNA and protein expression from mid-gestation onwards, our present findings are largely consistent with those of previous reports. Using northern blot analysis, á2A adrenoceptor mRNA levels in whole rat brain have been found to be high at birth, and to decline during postnatal development to low adult levels.17,40 In contrast, [3H]clonidine binding to á2 adrenoceptor protein increases during the postnatal period.23,43 Since [3H]clonidine labels other á2 adrenoceptor subtypes, in addition to á2A, it is not clear which subtype this increased postnatal labeling represents. However, consistent with our present findings, there is significant [3H]clonidine labeling of á2 adrenoceptor pro-

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tein at birth, a time at which there is little expression of á2B or á2C mRNA or protein (see accompanying paper).71,72 At this age, [3H]clonidine binding is high in regions such as the hippocampus and neocortex,23 which we have found to exhibit high levels of [3H]idazoxan binding. Whereas we have identified á2A adrenoceptor mRNA and binding sites during the prenatal period, we do not yet have evidence as to whether these sites are functional. Their early transient appearance, at a time which coincides with the arrival of NE afferents,57,62 would suggest a specific functional role for this receptor type in neuronal ontogeny. Electrophysiological data indicate that LC neurons are active during the prenatal period,44 and lesion studies suggest a possible neurotrophic role for brain NE, particularly with regard to regulation of neuronal differentiation (reviewed by Leslie32). However, the receptor type involved has not yet been identified. Although the â receptor has been implicated in modulating physiological plasticity and metabolism in developing brain,60,61 our present data suggest that á2A adrenoceptors may also be important mediators of NE effects during ontogeny. Whereas there are some preliminary data which suggest that á2 receptors may regulate neuronal differentiation in developing Xenopus oocytes and rat brain,55,69 more specific pharmacological studies will be required to confirm this hypothesis. The transient expression of á2A adrenoceptor mRNA and protein in the parietal cortex during the perinatal period may have particular functional relevance, given previous findings that LC neurons are extremely sensitive to somatosensory stimuli at this time.44,50 Receptor expression is up-regulated in the parietal cortex from E19 until P1, when cells destined for layers II–IV are migrating into the cortical plate.4 During the same period, LC neurons are not spontaneously active but are strongly responsive to noxious and non-noxious sensory stimuli.44 This unique perinatal sensitivity to sensory stimuli, which declines after the first postnatal week, may result from transient electrotonic coupling of LC cells and the transient functional appearance of an excitatory á1 autoreceptor.39 The up-regulated expression of á2A adrenoceptors in the fetal somatosensory cortex, coupled with the high sensitivity of LC neurons to sensory stimuli during this period, may indicate a specific role of NE in the anatomical and functional maturation of this brain region. NE has also been shown to play a role in the development of early odor preference in infant rats, induced by a combination of odor and tactile stimulation.50,64 During the critical perinatal period when odor preference is acquired,73 several olfactory structures in the brain express moderate to high levels of á2A receptor mRNA, including the olfactory bulb, anterior olfactory nucleus, piriform cortex, lateral olfactory tract nucleus and endopiriform nucleus. Especially striking is the transient up-regulation in

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the lateral olfactory tract nucleus, where the decline of á2A mRNA expression after the first postnatal week corresponds to the end of the critical period in associative olfactory learning. Thus, although â adrenoceptors have been primarily implicated in the acquisition of learned olfactory preferences in rat pups,50,63 our present anatomical data suggest that á2A receptors may also play a role in the development of olfactory function. CONCLUSION

Our present anatomical data indicate that there is an early developmental expression of á2A adrenoceptors both in central noradrenergic neurons and their target cells. Whereas the presynaptic expression

remains constant throughout development, there is an early postnatal decline of á2A receptor expression in many other brain regions. The transient perinatal expression of this adrenoceptor type, which coincides with a period of LC hyperreactivity to sensory stimuli, may indicate a specific functional role for the á2A receptor in the developing brain. The pattern of widespread, transient expression of á2A receptors in the fetal brain is in marked contrast to the postnatal development of the á2C receptor type, as described in the accompanying paper.72,18not in text

Acknowledgements—This work was supported by PHS grant nos DC 00450 and NS 30109. We would like to thank Dr K. Lynch for providing the á2A cDNA.

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