Neuroscience Letters, 120 (1990) 97-100 97 Elsevier Scientific Publishers Ireland Ltd.

NSL 07330

Autoradiographic localization of a2-adrenoceptors in chick brain

Arsenio Fernf indez-L6pez l, Ca rmen del Arco 3, An t o n i o M. Gonzfilez 3, Tomfis G 6 m e z 1, Pedro Calvo 2 and Angel Pazos 3

Departments of tCellular Biology and Anatomy, and 2Biochemistry and Molecular Biology, University of Lebn, Lebn (Spain) and 3Department of Physiology and Pharmacology, University of Cantabria, Santander (Spain)

(Received 10 May 1990; Revised version received 27 July 1990; Accepted 14 August 1990)

Key words: [3H]UK 14304; Autoradiography; ~t2-Adrenoceptor; Chick brain; Hyperstriatum; Visual pathway

ct2-Adrenoceptors were localized in the chick brain by 'in vitro' receptor autoradiography using pH]UK 14304 as a ligand. High or very high densities of binding sites were found in the hyperstriatum, tuberculum olfactorium, hypothalamic nuclei, rectum opticum and some medullary nuclei. Comparatively, intermediate densities were observed over the thalamie nuclei and locus ceruleus, among others. Low densities of et2-adrenoeeptors were detected in the paleostriatum, hippocampus and cerebellum. Our data indicate that at2-adrenoceptors in the chick brain present similar proper- ties and homologous anatomical distribution to those reported in mammalian brain.

The existence of two types of noradrenergic receptors, ct and fl, is widely supported by functional, pharmacolo- gical and biochemical data [1]. In addition, several sub- types of these receptors have been identified (~q, ~t2, ill, fiE) [7]. Brain 0t2-adrenoceptors are of special interest, since most of them appear to be presynaptic autorecep- tors, playing a role in the regulation of the release of nor- adrenaline [9]. The pharmacological properties and ana- tomical distribution of this receptor subtype have been studied in detail in rat and human brain, using [3H] para- aminoclonidine [14, 17] and [3H]UK 14304 [10, 12].

The existence of several neurotransmitters, as well as some presynaptic aspects of their activity, have been well studied in the brain of non-mammalian vertebrates [2, 6, 8-13]. In contrast, little information is available on the presence, distribution and characteristics of neurotrans- mitter receptors [4, 5]. In this sense, identification of neurotransmitter receptors can be used as a tool for the analysis of the phylogeny of chemical transmission, and the knowledge of their distribution can contribute to establish the homologies between different neuronal nu- clei among vertebrates, as well as to differentiate neural populations. The development of a variety of histochem- ical techniques has in fact allowed a better understand- ing of comparative brain organization [15]. Thus, the use of ' in vitro' autoradiographic procedures allows a micro-

Correspondence: A. Pazos, Dept. of Physiology and Pharmacology, Unit of Pharmacology, University of Cantabria, 39011, Santander, Spain.

scopic analysis of the distribution of neurotransmitter receptors.

In the case of ~t2-adrenoceptors, their detailed distribu- tion in the avian brain remains unknown. Information about this distribution could also improve our know- ledge of the functional relevance of noradrenergic sys- tems in avian brain. In this regard, we present herein a quantitative autoradiographic study on the localization of ~t2-adrenoceptors, labeled with [3H]UK 14304 (5- bromo-6[2-imidazolin-2-ylamino]quinoxaline), in chick brain.

Tissues of 4 male chicks (Gallus gallus), 350-400 g, were used. Ten gm coronal sections from several brain levels were mounted onto gelatin-coated slides and stored at -20°C until used. After a 15 min preincuba- tion at room temperature in Tris-HCl buffer (50 mM, pH 7.7) containing 0.1 mM MnCI2, tissue sections were incubated with 5 nM [3H]UK 14304 (82.7 Ci/mmol, New England Nuclear) for 90 min under the same conditions. Following the incubation, sections were washed for 5 min in ice-cold buffer and then dried in cold air stream. Non-specific binding (10-20% of the total binding) was defined as that remaining in the presence of 10/zM phen- tolamine [12]. Sections were exposed at 4°C for 8 weeks to tritium-sensitive film. The autoradiograms were ana- lyzed and quantified using an IBAS computer-assisted image analysis system. Appropriate standards [16] exposed together with the tissues, allowed the transfor- mation of densitometric reading, obtained after several measurements in each nucleus, into receptor densities (fmol/mg protein).

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The b inding of [3H]UK 14304 to moun ted sections of

chick bra in was reversible and saturable, the KD value

being 1.2 + 0.75 nM. The profile of affinities of several

compounds for [3H]UK 14304 b inding to sections met

the criteria for ~2-adrenoceptors (clonidine, phentola-

mine > adrenal ine > yohimbine > > prazosin > propra-

nolol) (data not shown).

The autoradiographic localization of [3H]UK 14304

Fig. 1. Autoradiographic localization of pH]UK 14304 (5 nM) binding sites at different levels of chick brain. Dark areas represent structures with high densities of binding sites. Coronal sections through the septum (A, B), anterior and posterior hypothalamus (C, D), upper and lower midbrain (E, F) and medulla (G). A, C-G: total binding of [3H]UK 14304; B: binding in the presence of 10 -5 M phentolamine. Note the high concentrations of ct2-adrenoceptors in areas such as the hyperstriatum (H), ventrale and dorsale (HV, HD), hypothalamus (Hy), griseum periventriculare (GPV), tectum opticum (T), specially over layer III (III), locus ceruleus (LC) and nucleus vestibularis medialis (NVM). In contrast, paleostriatum augmenta-

tum (PA), neostriatum (N), cortex (Cx) and cerebellum (Cb), among other structures, were poor in these sites. Bar = 0.2 cm.

binding sites throughout the chick brain was heteroge- neous (Fig. 1, Table I).

In chick telencephalon, very high densities of autora- diographic grains were seen in nucleus accumbens, sep- tum, hyperstriatum dorsalis and ventrale, and some neostriatal areas. Structures with comparatively high densities included tuberculum olfactorium and lobus paraolfactorius. The hyperstriatum accessorius con- tained an intermediate level of [3H]UK 14304 binding while low amounts of autoradiographic grains were observed over paleostriatum augrnentatum, neostriatum lateralis, cortex and hippocampus (Fig. 1A-C). In dien- cephalon, the hypothalamic nuclei showed very high densities of [3H]UK 14304 specific binding while thala- mic nuclei were moderately rich (Fig. 1C, D). In mesen- cephalon, high densities of ~2-adrenoceptors were found in griseum periventriculare, griseum centrale, and the stratum griseum centrale of the tectum opticum, while intermediate densities were found in the striatum gri- seum superficiale of the rectum opticum and locus ceru- leus (Fig. 1E, F). The cerebellum contained a very low level of 0t2-adrenoceptors (Fig. 1E, F). In contrast, in medulla, the nucleus vestibularis medialis and nucleus of the vagus presented high densities, the remaining areas being poor in [3H]UK 14304-specific binding (Fig. 1E- G).

The main conclusion of our study is that the chick brain contains 0t2-adrenoceptors; according to the profile of affinities, they appear to belong to the ~t2A subclass [3]. Furthermore, their distribution is highly heterogeneous and, in general terms, it correlates well with that found in mammalian brain. In this regard, while the paleostria- tum augmentatum and paleostriatum primitivum showed low or very low levels of specific binding, high or very high autoradiographic densities were found in neostriatum and hyperstriatum. These data are in agree- ment with the relevant presence of ~2-adrenoceptors in rat and human neocortex and the low densities found in basal ganglia in both species [10, 12, 17]. This correlation supports the proposed homology between paleostriatum augrnentatum and primitivum and mammalian striatum and pallidum, respectively [11]. Furthermore, neostria- tum and hyperstriatum appear to be comparable to neo- cortex in mammals [11, 15]. In a similar way, the hypo- thalamus, olfactory areas, and motor nucleus of the vagus also contained high or very high densities of ~2- adrenoceptors in both chick and rat brain. However, the parallelism was not complete. For example, in rat hippo- campus, a high density of ~2-adrenoceptors is observed over the stratum lacunosum molecular of CA 1 [17]. This pattern of labeling was not found in chick hippocampus.

By using fluorescence microscope techniques, adrener- gic innervation has been described in avian brain areas

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such as septum, dorsal thalamus, hypothalamus and mesencephalon [2, 6, 8, 13]. This distribution correlates well with that of ~2-adrenoceptors described above.

Our results might suggest a role for ~2-adrenoceptors in the regulation of several central functions in the avian brain. Thus, the presence of adrenergic innervation and [3H]UK 14304 binding in the hypothalamus could sug- gest an involvement of this system in the activity of the hypothalamic-hypophyseal axis. Similarly to the mam- mals, ct2-adrenoceptors appear to play an important role in the avian visual and olfactory systems, since high densities of labelling are present in areas involved in those pathways [11], such as the hyperstriatum dorsale and ventrale, tectum opticum, dorsal thalamus and tuberculum olfactorium of the chick. Moreover, the presence of important densities of [3H]UK 14304 bind- ing sites over the motor nucleus of the vagus in the chick may be the substrate for the reported cardiovascular ac- tions of ~2-noradrenergic agents, as it has been discussed for the mammalian brain [17].

In conclusion, our data suggest that ~2-adrenoceptors,

TABLE I

DENSITIES OF [3H]UK 14304 BINDING IN THE CHICK BRAIN

The densities correspond to 5 nM [3H]UK 14304 and are expressed as mean + S.D.

Brain region fmol/mg prot.

Tuberculum olfactorium 576 4-12 Nucleus accumbens 624 4-16 Paleostriatum augmentatum 150 + 18 Septum 676 __+ 20 Hyperstriatum accessorius 401 4- 10 Hyperstriatum dorsale 636 + 7 Hyperstriatum ventrale 653 4- 11 Medial neostriatum 450 4- 18 Intermedio-ventral neostriatum 596 4- 28 Hippocampus 92 + 15 Cortex 291 4- 19 Medial hypothalamus 827 + 3 Lateral hypothalamus 774 4- 20 Nucleus dorsalis thalami 540 4-10 Griseum periventriculare 581 4- 7 Griseum centrale 427 4- 20 Tectum opticum

stratum opticum (layer I) 363 4-10 stratum griseum et fibrosum superficiale (layer II) 353 + 11 stratum griseum centrale (layer III) 477 + 12

Nucleus locus coeruleus 402 4- 40 Cerebellum 22 4- 23 Nucleus pontis medialis 282 4- 37 Nucleus vestibularis medialis 523 4- 8 Nucleus vestibularis spinalis 174 4- 11 Nucleus motor of the vagus 481 4-21

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as labeled with [3H]UK 14304, could be functionally relevant in the avian brain.

The technical equipment used in this work was partial- ly supported by a DGICYT grant, Ministry of Educa- tion (PB87/1038). We are most grateful to Mr. J.M. Maniega and Ms. E. Lop6z-Mendieta (Univ. Lebn) for their technical assistance and Dr. J. Tolivia (Univ. Oviedo) for critical comments. A.M.G. is a recipient of a 'Fondo de Investigaciones Sanitarias' predoctoral re- search fellowship.

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