Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain

Pergamon

PII:

Neuroscience Vol. 77, No. 1, pp. 247–255, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00424-1

DISTRIBUTION OF [3H]DIADENOSINE TETRAPHOSPHATE BINDING SITES IN RAT BRAIN F. RODRIuGUEZ-PASCUAL,* R. CORTE ´ S,† M. TORRES,* J. M. PALACIOS‡ and M. T. MIRAS-PORTUGAL*§ *Departamento de Bioquı´mica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain †Departamento de Neuroquı´mica, Centro de Investigacio´n y Desarrollo, CSIC Jordi Girona 18–26, 08034 Barcelona, Spain ‡Laboratorios Almirall, Research Center, Cardener 68, 08024 Barcelona, Spain Abstract––The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation. The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. Copyright ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: diadenosine tetraphosphate, Ap4A binding sites, purinergic receptors, autoradiography, rat brain, central nervous system.

Cellular signalling via ATP and other nucleotides has received increased attention over the last years and, in spite of the difficult methodological approaches, the family of P2 purinoceptors is a rich area for research and debate.1,7,12 The presence of diadenosine polyphosphates (tetra-, penta- and hexaphosphates) in secretory granules co-stored with ATP, as well as their joint release in a calcium-dependent manner after stimulation, enlarge the number of natural nucleotidic compounds able to achieve an extracellular signalling function.26,28,32,34,36 Moreover, the existence of veryhigh- and high-affinity binding sites in neural preparations, with Kd values being respectively under the nanomolar and in the micromolar range, strongly supports the role of diadenosine polyphosphates (ApnAs) in extracellular communication.25,29,33 The structural similarities between ATP and the ApnAs raise the question about the existence of common or independent receptors, and the problem is far from being solved. Single-cell studies with §To whom correspondence should be addressed. Abbreviations: Ap2A, diadenosine diphosphate; Ap4A, diadenosine tetraphosphate; ApnA, diadenosine polyphosphate; á,â-CH2-ATP, á,â-methylene ATP; HEPES, N-2-hydroxyethylpiperazine-N*-2-ethanesulphonic acid; HPLC, high-performance liquid chromatography; TBA, tetrabutylammonium.

endothelial cells from bovine adrenal medulla show that some metabotropic receptors able to mobilize calcium from internal stores respond to both substances.9 Also, a potent activation of diadenosine tetraphosphate (Ap4A) on a cloned P2u purinoceptor from a human astrocytoma cell line has been reported.18 Nevertheless, this is not the case for noradrenergic chromaffin cells, where the ionotropic ATP receptor does not respond to ApnAs.8 A more complex picture exists in rat and guinea-pig brain, where specific receptors for ApnA, able to induce in synaptic terminals a calcium entry not crossdesensitized by ATP and analogues, have been demonstrated.27,30 The existence of ionotropic receptors activated by both ATP and ApnA compounds has also been reported in rat sensory neurons.17 For a rational approach to the problem of the specific distribution of receptors in neural tissues, the development of specific and non-hydrolysable ATP and ApnA analogues, and the cloning and expression of different members of the P2 purinoceptors are necessary. To date, with the tools available, the distribution of P2x purinoceptors in rat brain and spinal cord has been characterized by autoradiographic studies of the binding of the analogue [3H]á,â-methylene-ATP ([3H]á,â-CH2-ATP), and a widespread distribution has been found.5 The cloning of three different P2x purinoceptors from vas

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Fig. 1. Two-step chromatographic purification of [3H]Ap4A. (A) Ion exchange chromatography of the reaction mixture on a QMA MemSep 1000 cartridge. The NH4HCO3 gradient and the radioactivity of a 3.5-µl aliquot from each fraction are shown. (B) Ion pair HPLC of the pooled fractions from step A. The absorbance (260 nm) profile and the radioactivity of a 4-µl aliquot from each fraction are shown. The UV detector attenuation was 0.001 a.u.f.s. (absorbance units full scale) and integrator attenuation was 256 m.f.s. (millivolts full scale). The conditions were as described in Experimental Procedures. The horizontal bar indicates the fractions pooled and the vertical bars the positions of mononucleotide and dinucleotide markers.

deferens, PC12 cells and sensory neurons was recently reported. Studies with Northern blotting for their respective mRNAs demonstrated that the PC12 purinoceptor (P2x2) was present in the brain and spinal cord, the vas deferens purinoceptor (P2x1) was present in the spinal cord, but not in brain in significant amounts, and the purinoceptor from sensory neurons (P2x3) presented a highly selective localization in nociceptive neurons.6,10,19,39 As Ap4A is a natural component of secretory vesicles, exhibiting very-high-affinity binding sites in neural preparations, we used [3H]Ap4A as a ligand to study, using autoradiography, the regional distribution of its binding sites in the brain. The binding sites present specific distribution areas, significantly different from those reported for P2x purinoceptor and adenosine. EXPERIMENTAL PROCEDURES

Synthesis and tetraphosphate

purification

of

[3H]diadenosine

Since the radioactively labelled ligand Ap4A is not commercially available, it was synthesized according to the procedure described by Ng and Orgel,21 and modified by Prescott and McLennan35 based on nucleotide condensation in the presence of carbodiimide. Initially, appropriate volumes of stock solutions of MgCl2, AMP and 2,8[3H]ATP were combined in an Eppendorf tube and dried under a gentle stream of nitrogen. One hundred microlitres of a solution of 2.5 M 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 1.7 M HEPES–NaOH (pH 6.8) were then

added to give 300 mM MgCl2, 300 mM AMP and 100 µM 2,8-[3H]ATP (310 µCi), and the residue was carefully dissolved. The tube was incubated at 37)C for 18 h, after which the reaction was terminated by dilution in 5 ml of 50 mM NH4HCO3 (pH 8.6). The mixture was then injected at a flow rate of 1 ml/min on to a QMA MemSep 1000 Ion Exchange Membrane Chromatography Cartridge (Millipore) equilibrated in 50 mM NH4HCO3 (pH 8.6). After elution of all unbound material from the cartridge by passing 15 ml of 50 mM NH4HCO3 (pH 8.6), nucleotides were eluted with a 20-ml linear gradient of 50 mM to 0.7 M NH4HCO3 (pH 8.6) and 1-ml fractions were collected. Portions of 3.5 µl of each fraction were added to 2 ml of scintillation liquid (Ready Safe, Beckman) and the radioactivity was determined in a LS3801 Beckman Counter. This first chromatographic step (Fig. 1A) was intended to separate the synthesized [3H]Ap4A from the larger possible amounts of AMP and diadenosine diphosphate (Ap2A), the other product of AMP condensation. The cartridge employed had a high capability of binding but a poor resolution, and it was highly appropriate for this purpose. The fractions corresponding to the elution time of Ap4A standard were combined, lyophilized and resuspended in 100 µl of 10 mM K2HPO4, 2 mM tetrabutylammonium (TBA) and 18% acetonitrile (pH 7.5). The final mixture was further purified by highperformance liquid chromatography (HPLC). The chromatographic system consisted of a Waters 600E delivery system working isocratically at a flow rate of 2 ml/min, a U6K injector, a 481 ëmax UV detector and a 745 data module integrator. In the performance of ion pair HPLC chromatography, the mobile phase was composed of 10 mM K2HPO4, 2 mM TBA and 18% acetonitrile (pH 7.5), with a Spheri-10 RP-18 (22 cm length, 0.46 cm i.d.) column from Brownlee (Applied Biosystem). Under these elution conditions, the first adenine nucleotides to elute were the mononucleotides AMP and ADP, followed by the adenine

Diadenosine tetraphosphate binding sites in rat brain dinucleotide Ap2A, which presented a retention time very close to ATP; Ap4A was the final nucleotide to elute, quite distant from the former compounds. Detection was at 260 nm and 1-ml fractions were collected (Fig. 1B). The radioactivity from 4 µl of each fraction was determined. A unique peak corresponding to the elution time of Ap4A standard and containing almost all the radioactivity was obtained. Finally, fractions containing pure [3H]Ap4A were combined, lyophilized and employed for the binding experiments. Preparations of animals and sections Eight male Wistar rats (160–180 g body weight; Iffa–Credo, Lyon, France) were used in this study. The animals were housed four per cage and kept in a controlled environment with a 12-h light/dark cycle and 22&2)C room temperature, and given free access to food and water. Animal care followed the Spanish legislation on ‘‘Protection of Animals Used in Experimental and Other Scientific Purposes’’, in agreement with the European regulation (O.J. of E.E. L35871 18/12/1986). The animals were decapitated, the brains and adrenal glands rapidly removed and frozen on dry ice. Sections (14 µm thick) were cut using a cryostat (2800 Frigocut, Reichert–Jung, Germany) and thawmounted on to microscope glass slides pretreated with 3-aminopropyltriethoxysilane. The sections were kept at "20)C until used. Ligand incubation conditions Dissociation and association experiments were carried out in order to determine the best washing and incubation times.23 Slides containing adrenal gland and sagittal brain sections were preincubated for 10 min at 4)C in 20 mM Tris–HCl buffer (pH 7.4) containing (in mM): NaCl 122, KCl 3.1, KH2PO4 0.4, NaHCO3 5 and MgSO4·7H2O 1.2. Subsequently, tissue sections were incubated in the same buffer in the presence of 1 nM [3H]Ap4A (specific activity 25.72 Ci/mmol) for 6 h at 4)C. The non-specific binding was determined in sections co-incubated with 10 µM unlabelled Ap4A. The slides were then washed in ice-cold buffer during periods of time ranging from 20 min to 2 h. The tissues were wiped off from the slides using glass microfibre filter disks and radioactivity measured in a liquid scintillation counter. Two washes of 10 min each were chosen for further experiments. In association experiments, sections from adrenal glands and brain were processed as described above, with varying incubation times (2, 6 and 12 h), followed by two 10-min washes. The tissues were wiped off from the slides and counted by scintillation. Maximal association was already achieved after 6 h incubation. Along the experimental time, controls for [3H]Ap4A hydrolysis were done by HPLC as described.37 Mapping experiments Rat brain sections from several representative coronal, sagittal and horizontal planes, together with sections from adrenal glands, were labelled according to the following final protocol: 10 min preincubation at 4)C, 6 h incubation with 1 nM [3H]Ap4A at 4)C and two 10-min washes at 4)C. Consecutive sections were incubated in the same conditions, but in the presence of 10 µM unlabelled Ap4A to generate the blanks. Finally, the tissues were rinsed in distilled water, quickly dried with cold air and exposed to 3H-sensitive film (Hyperfilm-3H, Amersham, U.K.), together with plastic 3H-standards (3H-Microscales, Amersham). After 13 months exposure at "20)C, the autoradiograms were developed with Kodak D19 and fixed. The exposed tissue sections were stained with Cresyl Violet.

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Quantification of the autoradiograms was performed with a computerized image analyser MCID-M4 (Imaging Research Inc., Ontario, Canada). Materials AMP, ADP, ATP, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and TBA were from Sigma. Ap4A was from Boehringer. 2,8-[3H]ATP (30.90 Ci/mmol) was from Amersham International. RESULTS

Yield and control of the [3H]diadenosine tetraphosphate ligand Ap4A was synthesized as described in Experimental Procedures, by condensation in the presence of carbodiimide of labelled [3H]ATP with a large amount of AMP (3000 times excess) in order to assure complete [3H]ATP condensation. The mixture also produced significant amounts of Ap2A, due to the condensation of two molecules of AMP. Its elution presented a retention time similar to that of ATP in the chromatographic conditions described here and did not interfere with [3H]Ap4A, which is produced in smaller amounts. The total radioactivity recovered from the first chromatographic step on QMA ion exchange chromatography was 53.68% with respect to the total [3H]ATP employed (Fig. 1A). Following this step there still remained significant amounts of other adenine nucleotides. A second step of purification was necessary and HPLC was used because of its capability of resolution. In Fig. 1B the presence of a well-defined and neat peak of labelled [3H]Ap4A is seen, suitable for the binding experiments. From the radioactivity and absorbance determinations, and from comparison with standards of Ap4A, a specific activity for the synthesized [3H]Ap4A of 25.72 Ci/mmol was calculated, with a yield for the overall process of about 43%. The specific activity was similar to that of the [3H]ATP employed in the reaction (30.90 Ci/mmol). [3H]Diadenosine tetraphosphate binding to brain sections Labelling conditions for autoradiography were selected in preliminary experiments where bound radioactivity was determined by counting wiped off tissues following different incubation conditions. Initial incubation conditions were chosen according to data obtained from rat brain synaptosomal preparations.25 Due to the specific activity of the labelled ligand, all of the tissue binding experiments were carried out to 1 nM concentration, 10 times the Kd value for the high-affinity binding sites in rat brain synaptic terminals.25 Although the ligand is slowly degraded by specific ectonucleotidases, when compared with the mononucleotides, significant degradation occurs at room temperature. To avoid this inconvenience, the

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Fig. 2. Time-course of association and dissociation of [3H]Ap4A from slide-mounted tissue sections. Slides containing sagittal brain sections were incubated with 1 nM [3H]Ap4A in the absence (specific) or presence (non-specific) of 10 µM unlabelled Ap4A. In association experiments (A), sections were incubated for periods of time ranging from 2 to 12 h, followed by two 10-min washes in ice-cold buffer. For dissociation experiments (B), the slides were incubated for 6 h with the ligand and then washed during different periods of time (from 10+10 to 60+60 min). The tissues were wiped off from the slides and counted by scintillation, as described in Experimental Procedures.

incubation of the sections was carried out at 4)C. The control of stability for the ligand was carried out using HPLC, as described for cellular and synaptosomal preparations.25,33 As the accessibility of the ligand to binding sites is very different from a synaptosomal preparation when compared with a tissue slice, it was necessary to pay special attention to the association equilibrium time and the ligand dissociation during the washings. Results are shown in Fig. 2. In dissociation experiments, a good ratio of total/ non-specific binding, together with a high level of specific binding, was obtained after two washes of 10 min each. The non-specific binding, defined as the binding remaining in the presence of 10 µM unlabelled Ap4A, was equal to background values in all brain sections examined. Thus, these conditions were selected for further experiments. Regional distribution of [3H]diadenosine tetraphosphate binding sites The Ap4A binding sites were widespread but heterogeneously distributed throughout the rat brain. The range of densities was from 3 to 15 fmol/mg tissue in gray matter areas, while white matter was devoid of specific binding, showing background levels. Co-incubation of [3H]Ap4A with an excess of unlabelled Ap4A (10 µM) resulted in autoradiographic images which were not different from film background (Fig. 4B). Table 1 and Figs. 3 and 4 summarize and illustrate the quantitative and qualitative distribution of Ap4A binding sites in the rat brain and adrenal gland. As illustrated, the highest densities of [3H]Ap4A binding sites were observed in the seventh cranial (facial) nerve, medial superior olive, pontine nuclei, glomerular and external

plexiform layers of the olfactory bulb, and granulle cell layer of the cerebellar cortex. Intermediate levels of binding were found in different cortical areas, including the neocortex and entorhinal cortex, olfactory tubercle, and several nuclei of the amygdala, thalamus and hypothalamus. The hippocampal formation also presented intermediate densities in the oriens and pyramidal layers. Lower densities were seen in many other brain areas, such as the basal ganglia (caudate–putamen, globus pallidus), midbrain and brainstem. In the midbrain, the central gray and the superficial gray layer of the superior colliculus were relatively enriched in [3H]Ap4A binding sites when compared to surrounding areas. Binding to the adrenal gland was done because neurochromaffin cells from the adrenal medulla were the first neural preparation where high-affinity binding sites for Ap4A were found. The adrenal medulla showed, as expected, a high density of binding sites, to an extent similar to the richest areas of the brain. In contrast, the lowest densities of specific binding were measured in the adrenal cortex. DISCUSSION

In the present report, the quantitative distribution of [3H]Ap4A binding sites has been studied in rat brain and adrenal gland. The quality of the autoradiograms obtained was limited by two factors: (i) the low specific activity of the ligand and (ii) the relatively low density of binding sites. Nevertheless, the results clearly show that Ap4A binding sites are distributed heterogeneously in the rat brain and adrenal gland, and below the detection level in white matter tracts.

Diadenosine tetraphosphate binding sites in rat brain

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Table 1. Quantitative regional distribution of [3H]diadenosine tetraphosphate binding sites in the rat brain and adrenal gland Region Olfactory bulb Glomerular and external plexiform layers Internal granular layer Cerebral cortex Frontal cortex Frontoparietal cortex Motor area Somatosensory area Striate cortex Entorhinal cortex Olfactory tubercle Caudate–putamen Globus pallidus Septum Thalamus Hypothalamus Amygdala

Mean&S.E.M. (n)

10.57&2.23(3) 5.57&0.97(3) 9.57&0.39(3) 8.70&0.28(6) 8.84&0.21(7) 7.84&0.11(6) 8.27&0.72(5) 8.13&0.65(6) 5.41&0.16(8) 5.91&0.52(4) 6.25&0.35(6) 7.66&0.23(8) 8.08&0.14(4) 9.74&0.04(3)

Region Hippocampus Dentate gyrus CA1, stratum oriens CA1, stratum radiatum Hilus (CA4) Cerebellum Granular layer Molecular layer Midbrain and brainstem Central gray Pontine nuclei Medial superior olive Facial nucleus Adrenal gland Cortex Medulla

Mean&S.E.M. (n)

6.13&0.10(6) 7.14&0.09(8) 5.59&0.19(6) 6.97&0.11(6) 11.46&0.18(8) 7.75&0.17(8) 5.45&0.09(6) 13.49&0.92(3) 13.22&0.15(3) 15.60&0.42(3) 2.94&0.11(6) 10.98&0.33(6)

Data are expressed as mean&S.E.M. Each value was calculated from 10 to 100 measurements depending on the size of the structure (n denotes the number of animals). Other brain structures with lower binding density have not been included.

As chromaffin cells were the first system where a specific binding for [3H]Ap4A was observed, special attention was paid to correlate the number of binding sites calculated from the isolated cells with respect to the adrenomedullary tissue. The result was encouraging because, taking into account that one million cells are approximately equivalent to 1 mg wet weight of tissue (0.1 mg of protein), the values for the binding sites were respectively 9 and 10 fmol/mg for isolated chromaffin cells and sections of the adrenal medulla.33 These results represent good support for the validity of quantification obtained for the different brain areas. In this way the quantity of specific binding sites obtained at 1 nM concentration, which is in the fmol/mg range of tissue in the section autoradiograms, correlates well with the Bmax obtained for the high-affinity binding sites (Kd=0.1 nM) in rat synaptic terminals25 and Torpedo synaptosomes.29 The physiological meaning of the brain binding sites needs to be analysed considering the concentration of these compounds in the extracellular media. An experimental approach to ApnA levels in isolated functioning synapses is not possible, but the brain perfusion of conscious rats indirectly allows it. In control animals the Ap4A levels are under 2 nM, the lowest concentration measurable, but drastic stimulation by amphetamine administration made the Ap4A levels reach maximal values of 15 nM in the perfusion samples.31 Thus, the enrichment of [3H]Ap4A binding sites quantified at 1 nM ligand concentration can be of particular interest, and should be studied thoroughly for a functional role of ApnAs. At present, a negative feedback for excitation in rat hippocampal slices has been described for ApnAs.16 In this area, specific binding in the CA1 and CA4 regions is reported here. Moreover, in synapto-

somes and isolated hippocampal neurons from rat brain, the ApnAs selectively potentiate N-type Ca2+ channels.24 One main question concerning the action of ApnAs is the specificity of the cellular responses compared with ATP, which physiologically is the most abundant. In the peripheral nervous system, the agonistic action of ApnAs on P2x, P2y and even P2u purinoceptors has been reported.2,9,14 In contrast, the ApnAs cannot activate the ionotropic ATP purinoceptor from noradrenergic chromaffin cells.8 Scarce but increasing data are available on the actions of ApnAs in the central nervous system; the opening of non-selective cation channels by ATP and Ap4A in nodose ganglion cells has been reported.17 The calcium entry induced by these compounds in rat midbrain and cerebellum synaptic terminals, not cross-desensitized by ATP and analogues, suggests the presence of different receptors for both types of compounds, at least in these areas.27,30 The presence of ectodiadenosine polyphosphate hydrolases in neural and vascular endothelial cells makes necessary a rigorous control of the integrity of the labelled ligand to avoid the presence of labelled ATP and derived nucleosides and nucleotides.13,22,37 As these enzymes exhibit affinity values in the 1–2 µM range (Km value), their catalytic centres are irrelevant as possible sites of binding at the 1 nM ligand concentration used in the present report. Recently, the distribution of binding sites for [3H]á,â-CH2-ATP, a ligand that preferentially binds to P2x purinoceptors, has been reported by two groups.3,5 The data of Bo and Burnstock5 show a widespread distribution of the binding sites, the nuclei of the thalamus, substantia nigra, hypothalamus and caudate–putamen being among the most densely labelled structures. These brain areas do not present

F. Rodrı´guez-Pascual et al.

Fig. 3.

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Diadenosine tetraphosphate binding sites in rat brain

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Fig. 4. Details from rat cerebellum and brainstem areas in sagittal sections labelled with [3H]Ap4A. (A) Autoradiogram control obtained as described in Experimental Procedures. (B) Consecutive brain sections incubated with addition of 10 µM unlabelled Ap4A to determine the background levels. (C) Cresyl Violet staining of the same section. Scale bar=1 mm (A=B=C).

a notorious [3H]Ap4A binding in the experimental conditions reported here, both compounds exhibiting very different pattern distributions in the autoradiograms. With respect to the results of Balcar et al.,3 the binding of [3H]Ap4A in our conditions is less by one order of magnitude when compared in fmol/ mg tissue. These results correlate well with the higher concentration of [3H]á,â-CH2-ATP employed (10 nM). It is noteworthy that, in autoradiography with [3H]á,â-CH2-ATP, the cerebellar cortex exhibits the highest density of binding sites, being susceptible of displacement by Ap4A to the extent of 70%. Regarding [3H]Ap4A binding, the facial nucleus, the pontine nuclei, the medial superior olive and the granular layer of the cerebellum present the highest densities of binding sites. The adenosine A1 and A2a receptors, which are found predominantly in the brain, have been studied

thoroughly, allowing a deeper comparative analysis. The regional distribution of [3H]Ap4A binding sites in the rat brain revealed some similarities and differences with respect to the autoradiographic localization of A1 adenosine receptors as labelled by [3H]cyclohexyladenosine.11 Both Ap4A and A1 sites were very abundant in the cerebellum; however, their laminar distributions clearly differed, since Ap4A sites were more enriched in the granule cell layer, whereas A1 receptors were mainly concentrated in the molecular layer. Relatively high densities of [3H]Ap4A and [3H]cyclohexyladenosine binding sites were found in the neocortex, thalamus and pontine nuclei, while structures such as the caudate–putamen and globus pallidus, among others, displayed low levels of binding of both ligands. On the other hand, different binding patterns of the A1 and Ap4A sites were observed in the entorhinal cortex (poor in A1

Abbreviations used in Figs 3 and 4 Acx Ame CA1 CG CPu DG Fr FrPaM gr Hip Hy

adrenal cortex adrenal medulla CA1 hippocampal field central gray caudate–putamen dentate gyrus frontal cortex frontoparietal cortex, motor area granule cell layer of the cerebellum hippocampus hypothalamus

mo MSO OBep OBgl OBig Pn Sp Str Th Tu 7

molecular layer of the cerebellum medial superior olive external plexiform layer of the olfactory bulb glomerular layer of the olfactory bulb internal granular layer of the olfactory bulb pontine nuclei septum striate cortex thalamus olfactory tubercle facial nucleus

Fig. 3. Distribution of [3H]Ap4A binding sites in the rat brain and adrenal gland. Autoradiograms were obtained from brain horizontal (A, C) and sagittal (E) sections, and the adrenal gland (G, H). Cresyl Violet staining of the corresponding tissue sections is shown in B, D and F. Strong labelling can be observed in the adrenal medulla, as well as in the pontine nuclei, medial superior olive, facial nucleus (7), granule cell layer of the cerebellum, and glomerular and external plexiform layers of the olfactory bulb. Scale bars=1 mm. (A=B=C=D=E=F=G; H).

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receptors and enriched in Ap4A sites) and the hippocampus, which contained very high densities of A1 receptors, with a laminar distribution very different from Ap4A sites. Even more significant are the differences with respect to A2a receptors, which are present in very restricted areas of the striatum, nucleus accumbens and olfactory tubercle. These receptors are expressed exclusively by the enkephalinergic striatal neuronal subpopulation that also selectively expresses the dopamine D2 receptor.15,20,38 Furthermore, no similarities have been found with respect to the adenosine transporter sites analysed by [3H]nitrobenzylthioinosine binding autoradiography.4 Apart from the comparative studies with other purinergic compounds, the [3H]Ap4A distribution of binding sites in the brain deserves special comments. This is the case of the facial nucleus, the most labelled structure, which receives the information from facial areas, including most of the taste sensorial inputs, through the seventh cranial nerve. After the facial nucleus, the most densely labelled structures correspond to the olfactory pathway. The receptor cells in the olfactory mucosa project into the olfactory bulb to the glomerular and external plexiform layers, where the synaptic connections with mitral and tufted cells are established. The axons from the olfactory bulb reach the olfactory tubercle, which also presents significant Ap4A binding sites. Following the functional pathways from the olfactory tubercle, the amygdala and the entorhinal area

of the cortex, which are components of the limbic system and are involved in affective behaviour, contain significant levels of [3H]Ap4A binding. Not only are the pathways for chemical senses densely labelled, but also the cerebellum and associated control movement areas. This is the case for the pontine nuclei, which is essential in cerebro-cerebellar connections, and for the medial superior olive. The axons from these nuclei reach, through the mossy fibres, the granular layer of the cerebellum. The presence of a high density of binding sites for [3H]Ap4A in the granular layer could be the result of pre- or postsynaptic receptors in this area. However, the technique as used here does not allow resolution to a cellular level.

CONCLUSION

The autoradiographic results support the existence of specific Ap4A binding sites. However, further work is necessary to elucidate the physiological role of ApnA compounds in specific areas of the CNS. Acknowledgements—We thank Erik Lundin for help in the preparation of the manuscript. This study was supported by grants from the Spanish Ministry of Education and Science CICYT (PB 92/230), from Biomed 2 no. PL 950676 from E.U. and from the Areces Foundation Neuroscience Programme. F.R.-P. was supported by a Fellowship from the Universidad Complutense.

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Diadenosine tetraphosphate binding sites in rat brain 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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