Brain adenosine receptors in Maudsley reactive and non-reactive rats

Brain Research, 421 (1987)69-74

69

Elsevier BRE 12866

Brain adenosine receptors in Maudsley reactive and non-reactive rats P.J. Marangos, T.R. Insel, P. Montgomery and E. Tamborska Unit on Neurochemistry, Biological Psychiatry Branch and Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 (U.S.A.)

(Accepted 17 February 1987) Key words: Adenosine receptor; Brain; Maudsleyrat; Autoradiography; Molecularlayer

Previous work in our laboratory has shown that the Maudsley reactive (MR) strain of rats cannot be differentiated from the Maudsleynon-reactive (MNR) strain regarding the number or affinityof their brain benzodiazepinebindingsites. In the present study we show that the number of cerebellar adenosine receptors (as studied using [3H]cyclohexyladenosine,[3H]CHA, as the iigand) are increased by 15-30% in the MR strain. This alteration was corroborated by quantitative autoradiographicanalysis and found to be localized to the molecular layer of the cerebellum where adenosine receptors are believed to reside on parallel fibers of cerebellar granule cells.

INTRODUCTION It has become increasingly evident that adenosine is a major non-peptide neuromodulator in brain. The purine adenosine and its metabolically stable analogs have been found to exert multiple effects on neuronal tissue. In brain slices adenosine inhibits the release of many different neurotransmitters 11,17. Adenosine has been demonstrated to inhibit neuronal firing primarily by inhibiting presynaptic excitatory transmitter release 17. This process, however, may be reversed by methylxanthines such as caffeine and theophylline which are potent and selective adenosine antagonists 2°. At the behavioral level, adenosine and particularly its metabolically stable analogs are potent sedatives 7,2° whereas caffeine, an adenosine antagonist, is a stimulant and convulsant at high doses 15'2°. At the biochemical level, adenosine is known to modulate adenylate cyclase activity, thereby influencing cyclic AMP levels 19. In a number of tissues this purine has a biphasic effect on cAMP production that is thought to be mediated by two types of

cell surface adenosine receptors termed AI (decreases cAMP) and A 2 (increases cAMP) 22'13. The actions of adenosine are thought to be mediated by specific cell surface receptors 19. Recently, these receptors have been characterized using labelled metabolically stable adenosine analogs such as [3H]cyclohexyladenosine, [3H]CHA and [3H]diethylphenylxanthine, [3H]DPX6,14j6. In an effort to further characterize the adenosine receptor and its relationship to behavior we investigated the receptor status in Maudsley reactive (MR) and Maudsley non-reactive (MNR) rats. These inbred Wistar rat strains, originally developed by Broadhurst 4, were initially selected for differences in defecation, presumably a measure of 'emotional reactivity'. Subsequent research has established that the MR strain also shows decreased exploration, longer avoidance latency, and decreased norepinephrine in peripheral tissue compared to their MNR congeners 5. In the present study we demonstrate that these strains also differ in the number of adenosine receptors in brain.

Correspondence: P.J. Marangos, Unit on Neurochemistry, Biological Psychiatry Branch, NIMH, Building 10, Room 3C-210, Bethesda, MD 20892, U.S.A.

70 MATERIALSAND METHODS Animals Pedigreed MR/N and MNR/N strains obtained from the NIH stock were raised and bred in our laboratory. The NIH stock of both strains was originally acquired by the National Institute of Health in 1963 at the 21st generation of inbreeding. These rats have been inbred for at least 40 subsequent generations at NIH without attenuation of the original behavioral differences 2. Animals were maintained in large plastic cages in a temperature controlled room with a 14 h light/10 h dark diurnal cycle. Pups were weaned at 21 days and reared in group cages with litter mates of the same sex. Behavioral testing The behavioral test apparatus was similar to that originally used by Broadhurst 4. The open field was a white-walled, circular arena, 1 meter in diameter placed on a Plexiglas base. The floor was marked off into concentric blocks of equal area for estimating locomotor activity. The open field was illuminated by a single photoflood suspended 36 in. above the floor. Room lights were extinguished to prevent the rat from seeing the observer. Tests were of 2-min duration. Each rat was weighed, then transported to the center of the open field for 10 s in a start box. Following removal of the start box, activity was assessed by the crossover of both front feet into a block. Fecal boli were counted at the end of each test. The open field was scrubbed with an ammonia soap solution and then dried between each trial. Each rat was used only one time. All testing was performed at least 3 days before sacrifice. Results of the behavioral testing on a representative subset of the animals utilized in this study are shown in Table I.

Membrane preparation After decapitation, brains were quickly removed and dissected into different regions on an ice-chilled glass plate. The brain was dissected according to the protocol of Glowinski and Iversen 8. All tissues were frozen and kept at -70 °C. Prior to use the tissues were thawed and homogenized in 25 vols. of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using the Brinkman Polytron (setting 5, 10 s). The resulting homogenate was centrifuged at 30,000 g for 20 rain and the pellet washed once more. The resulting membrane suspension was adjusted to a concentration of 2 units/ml with adenosine deaminase (Sigma Type III), incubated at 25 °C for 30 min followed by centrifugation (30,000 g for 20 min) and resuspended in 25 vols. of buffer. [3H ] C H A binding assay [3H]CHA binding assays were performed as previously described 16. In summary 0.2-0.6 mg of membrane protein was incubated with 4 nM [3H]CHA (25 Ci/mmol, New England Nuclear) in a final volume of 0.5 ml. Final buffer concentration was 50 mM Trischloride, pH 7.4. Non-specific binding was determined by incorporating 5/~M unlabeled C H A (Calbiochem) and routinely represented 20% of total binding at 4 nM [3H]CHA. The assay was incubated for 2 h at 25 °C and terminated by vacuum filtration on Whatman GF-B filters followed by three 5 ml washes with ice-cold buffer. Filtration was carried out using the Brandel Cell Harvester M-24R. The filters were air-dried and counted in 10 ml of Beckman Redi-Solv. [3H]CHA binding was performed using 2 nM of [3H]CHA for single point analysis and Scatchard analysis concentrations of [3H]CHA ranging from 0.25 nM to 24 nM were employed. A utoradiography mapping After decapitation brains were quickly removed

TABLE I Behavioral comparison of Maudsley reactive (MR) and Maudsley non-reactive (MNR) rats in open field test The indicated number of animals were tested as described in methods. Results indicate means +- S.E.M. Strain

n

Body weight (g)

Boli

Inner squares

Outer squares

% lnner squares

MR MNR

16 18

240 + 15.7 219.3 + 12.9

4.1 _+0.6 0.1 + 0.1"*

9,3 + 1.4 26,7 + 3.8**

25.0 + 4.0 27.9 _+3.4

29.4 + 2.9 47.8 + 3.3**

**P ~<0.01, Student's t-test.

71 and frozen on dry ice. They were stored a t - 7 0 °C until sectioning at - 1 4 °C in a cryostat (Harris, Billerica, MA). Sections of 16/~m thickness were then mounted onto chrome-alum covered slides. Adjacent sections were incubated in 50 mM Tris-HCl pH 7.5 with 1 nM [3H]CHA at 25 °C for2 h. Non-specific binding was assessed in a parallel series of slides incubated under identical conditions along with 10-6 M phenylisopropyladenosine (PIA). All slides were washed twice for 5 min in 50 mM Tris-HCl at 25 °C and then dried under a stream of cool air. Slides were apposed to ultrafilm (LKB) in X-ray cassettes for 2 weeks prior to development in D-19 at 22 °C for 4 min. Analysis of binding used a computer based system (Loats Assoc., Westminster, MD) for digitizing the autoradiographic image and tritium standards (Amersham) for converting optical density to fmol/mg protein values. Of key importance to this experiment was the consistent matching of an MR with an MNR brain. Brains were cut side-by-side to match orientation for sectioning and slides with sections from an MR brain were incubated and washed back to back in plastic iars (cytomailers) with the corresponding slides for MNR sections. Statistical analysis Data were statistically evaluated by using the unpaired Student's t-test analysis (two-tailed).

TABLE II Specific binding of [sH]CHA in various brain areas in Maudsley non-reactive (MNR) and Maudsley reactive (MR) rats The results shown are those of one experiment where the number of animals in each group was eight. This experiment was repeated twice with very similar results. Other brain areas tested which show no significant difference in [3H]CHA binding were hippocampus, striatum, and brainstem. Brain areas

Specific [3H]CHA binding (fmol/mg protein)

Cerebral cortex Cerebellum

MNR

MR

222.9 __-4.8 275.4 + 9.1

221.0 -+ 5.4 323.8 +_10.7"

*P < 0.005, Student's t-test.

periment) were examined for [3H]CHA binding capacity and the results obtained for one such experiment are shown in Table II. The number of binding sites observed with 4 nM [3H]CHA is significantly increased in cerebellar membranes with no change observed in the cerebral cortex. In all 3 experiments the increases observed in the MR cerebellar membranes ranged from 15 to 30%, each time significant at the P < 0.01 level. The single point data presented in Table II does not address the question of whether the number or the affinity of the adenosine receptors differ between

RESULTS SCATCHARD ANALYSIS OF [3H I CHA BINDING IN CEREBELLUM OF MNR AND MR RATS

Adenosine receptor binding Initial studies revealed that adenosine receptor binding parameters in the Maudsley strains were quite similar to those obtained in other rat strains 14. The binding of the adenosine agonist [3H]CHA was biphasic in nature with an approximate 2:1 ratio of low affinity (K d = 4.8 nM) to high affinity (K d = 0.5 nM) sites. The relative distribution of adenosine receptors in 7 brain areas was also quite similar to that observed in other rat strains 16. Preliminary analysis of adenosine receptor status in MR and MNR rats revealed that the cerebellum was selectively affected with no significant effects seen in other brain areas. This prompted us to focus on the cerebellum and one other brain area which we arbitrarily chose as the cerebral cortex in further studies. All 3 paired groups (n = 8 pairs in each ex-

0600

O ' ~ MNR HMR

0500

~ 0,400

m~0300

0.200

01001

|~ t

0

•

,,,, 50

100

o

150

200

250

300

350

400

450

BOUND (fmol/mg protein)

Fig. I. Scatchard analysis of [3H]CBA binding in cerebellum of MNR and MR rats. Aliquots of cerebellar membranes from 8 MR (0-0-0) and 8 MNR (C)-O-C)) rats were pooled and each membrane pool was titrated with [3H]CHA as described in methods. This experiment was repeated on one other pooled group with similar results.

72 TABLE III Kinetic parameters for [3H]CHA binding in the cerebellum of MNR and MR rats Cerebellar membranes from 8 MRN and 8 MR rats were pooled and subjected to Scatchard analysis. The binding parameters observed for both the high and low affinity site are shown. This experiment was repeated with similar results.

MNR MR

K d (nM)

Bm~ (fmol/ mg protein)

0.65 4.25 0.60 3.95

63 244 80 344

The autoradiographic results support and extend the binding data in that the only region where increased [3H]CHA binding sites are o b s e r v e d is in the molecular cell layer of the cerebellum in the M R strain. Previous autoradiographic studies from ours 1'12 and other 9'1° laboratories have shown that adenosine receptors are localized in the molecular cell layer of the cerebellum and p r o b a b l y reside on the parallel fibers of excitatory granule cells 1°. Fig. 2 illustrates the autoradiographic patterns routinely o b t a i n e d when M R and M N R cerebellar sections are directly compared. DISCUSSION

the groups. To answer this question we p o o l e d aliquots of cerebellar m e m b r a n e s from 8 M R and M N R rats. Scatchard analysis of each p o o l e d m e m b r a n e fraction was p e r f o r m e d with the result of a representative e x p e r i m e n t shown in Fig. 1. It is quite app a r e n t that the n u m b e r of cerebellar binding sites increases for both the high and low affinity adenosine agonist r e c e p t o r while the binding affinities are relatively unchanged. The respective binding p a r a m eters o b s e r v e d in this e x p e r i m e n t are shown in Table III. This analysis was p e r f o r m e d in one o t h e r paired group of animals with very similar results obtained.

The present r e p o r t shows by both ligand binding to m e m b r a n e p r e p a r a t i o n s and slide-mounted brain slices that adenosine receptors are specifically increased in the M R rat cerebellar molecular cell layer. Since previous work has shown that these adenosine receptors are on the granule cell parallel fibers 9 it is intriguing to speculate that the m o d u l a t o r y mechanisms of these excitatory glutamate utilizing cells are altered in the M R strain and that this m a y explain some of their behavioral properties. O u r results also predict that further studies related to cerebellar glutamatergic p a r a m e t e r s in M R rats would be of inter-

A utoradiographic studies To further substantiate and localize the strain differences in m e m b r a n e binding shown in Table II, analysis of [3H]CHA binding to slide m o u n t e d brain sections from M R and M N R rats were p e r f o r m e d with the densitometric results shown in Table IV.

est. Differences in binding assessed by r e c e p t o r autoradiography with tritiated ligands are generally suspect because of the potential for quench artifacts. Differential quenching in the M R and M N R cerebeilar sections could reflect differences in tissue density but this seems an unlikely explanation for the find-

TABLE IV Paired comparisons of MR + MNR autoradiographic binding of [3H]CHA (means + S. E.M. shown) The autoradiographic analysis was performed as described in methods.

Cortex Hippocampus Polymorphie layer Molecular layer Dorsal latera!:septum Caudate-putamen Cerebellum total Molecular layer Granule layer

n pairs

MR fmol/mg protein MNR fmol/mg protein % DIFF

t-values**

P

5

128.4 +_8.1

2.0298

NS

3 3 4 4 4 4 4

201.5 + 24.8 274.9 + 40.8 139.9 + 4.5 116.2 + 10.4 181.8 + 20.4 319,6 + 36.1 95.5 + 6.6

-1.2234 0.1241 1.5296 0.3077 1.4244 2.8029 1.7896

NS NS NS NS NS 0.05 NS

118 ___12.0 235.6 + 51.8 272.7 _+58.0 128.0 + 11,8 114.6 + 9.03 155.3 + 27.6 269.6 + 47.7 84.1 + 7.4

10.7 _+4.8 0.3 + 0.2 3.5 _+6.4 11.3 + 7,5 1.5 + 4.6 27.8 + 22.4 26.2 + 15.6 15.3 + 8.7

* This refers to the percent difference calculated for each MR-MNR pair with the mean percent difference across all pairs shown. ** Student's t-values calculated from paired data and not from the group means.

73

Fig. 2. Representative dark-field autoradiographic images of [3H]CHA binding in MNR (A) and MR (B) rat cerebellum. In both strains the binding is localized to the molecular cell layer with the MR strain exhibiting significantly more intense labelling.

74 ings reported here because nearly identical differ-

is required in order to substantiate either of those

ences were found with binding to m e m b r a n e homogenates, for which quench is not a factor.

speculations. Previous studies in our laboratory have also shown that various chronic stress paradigms can result in increased n u m b e r s of adenosine receptors 3

The adenosine system in brain is thought to mediate properties such as sedation with an increased tone of this system probably resulting in a decreased arousal state 7. O n e might, therefore, expect the M R

and it is possible that this may relate to the findings of the present study. A previous preliminary report TM showed that ben-

trait to be associated with decreased adenosinergic tone. A n explanation for our finding of increased

zodiazepine receptors were altered in M R rats. Recent work in our laboratory has, however, failed to

numbers of adenosine receptors might be that the

confirm this report and has shown that both central

M R trait may be associated with impaired coupling of the adenosine receptor to the effector system (cAMP

and peripheral type benzodiazepine receptors are not changed 2~. It is, therefore, possible that in-

generating) or with decreased adenosine levels which would likely produce both hyperarousal and in-

creased cerebellar adenosine receptors may be a rather selective neurochemical correlate of the M R trait.

creased levels of adenosine receptors. Further work

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