0031-3998/00/4802-0177 PEDIATRIC RESEARCH Vol. 48, No. 2, 2000 Copyright © 2000 International Pediatric Research Foundation, Inc. Printed in U.S.A. Maternal Caffeine Intake Has Minor Effects on Adenosine Receptor Ontogeny in the Rat Brain ULRIKA ÅDE´ N, ERIC HERLENIUS, LIE-QI TANG, AND BERTIL B. FREDHOLM Department of Physiology and Pharmacology, Karolinska Institute, S-171 77, Stockholm, Sweden [U.Å., B.B.F.]; and Department of Woman and Child Health, Astrid Lindgren Childen’s Hospital, S-171 76, Stockholm, Sweden [U.Å., E.H., L.-Q.T.] ABSTRACT Maternal caffeine intake has been suggested to influence the at any stage during development. [3H]-flunitrazepam binding, offspring. We have studied the effects of maternal caffeine intake representing GABAA receptors, showed large regional variations on adenosine and GABA receptors, targets for caffeine, during during ontogeny, but there were no clear differences between the development of the rat brain. Caffeine (0.3 g/L) was added to the caffeine-exposed and the nonexposed pups. Thus, exposure to a drinking water of rat dams during pregnancy and early postnatal low dose of caffeine during gestation and postnatal life had only life. Adenosine A1 and A2A and GABAA receptor development minor effects on development of adenosine A1 and A2A receptors was studied using receptor autoradiography and in situ hybrid- and GABAA receptors in the rat brain. (Pediatr Res 48: 177–183, ization. Pups were examined on embryonic d 14 (E14), E18, E21, 2000) 2 h after birth (P2h), P24h, postnatal d 3 (P3), P7, P14, and P21. Adenosine A1 receptor mRNA was detected at E14 and receptors Abbreviations at E18. A1 mRNA levels increased from the level reached at E18 E, embryonic day between P3 and P14 (maximally a doubling), whereas A1 recep- P, hours (h) or days after birth tors, studied by [3H]-1,3-dipropyl-8-cyclopentyl xanthine bind- DPCPX, 1,3-dipropyl-8-cyclopentyl xanthine ing, increased later and to a much larger extent (about 10-fold) CGS 21680, 2-[p-(2-carbonylethyl)phenylethylamino]-5Ј-N- postnatally. Caffeine treatment had no significant effect on aden- ethylcarboxamidoadenosine osine A1 receptors or on A1 receptor mRNA. A2A mRNA had SCH 58261, 5-amino-7-(2-phenylethyl)2-(2-furyl)-pyrazolo- reached adult levels by E18, whereas receptor levels were low or [4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine undetectable before birth and increased dramatically until P14. R-PIA, R-N6-phenylisopropyladenosine Ј Caffeine did not influence A2A receptors or A2A receptor mRNA NECA, 5 -N-ethylcarboxamidoadenosine Caffeine is the most widely consumed psychoactive sub- The only known biochemical effect of caffeine in the brain in stance. Whereas the use of alcohol and tobacco are usually concentrations relevant to daily intake of coffee is blockade of limited during pregnancy, beverages containing caffeine are adenosine A1 and A2A receptors (8). Both A1 and A2A recep- consumed at a normal or near normal rate (1). Negative effects tors are present at birth in the rat (9–11), but the major on fertility and birthweight, risk for prematurity, and congen- development in terms of density and coupling to second messen- ital malformations have been demonstrated in offspring of ger-forming systems occurs postnatally (12, 13). Secondarily to animals given high doses of caffeine (for review see Ref. 2), actions on adenosine receptors there are effects on dopaminergic but there is no conclusive evidence that normal human con- transmission (for review see Ref. 14, 15) and it is known that sumption has teratological consequences. Epidemiologic stud- alterations in dopaminergic transmission may result in develop- ies have shown a dose-dependent reduction in birth weight mental changes (16). In addition, there is evidence that caffeine when mothers consume more than seven cups of coffee a day can directly and indirectly influence GABAergic neurotransmis- (3, 4). Prenatal caffeine intake has been described to result in sion (8, 15). Studies on the effects of methylxanthines on benzo- behavioral hyperactivity in developing rodents (5–7). diazepine receptors are contradictory. An increased number of benzodiazepine binding sites in the adult mouse brain after Received December 6, 1999; accepted January 18, 2000. chronic administration of a high dose of caffeine has been reported Correspondence to: Ulrika Åde´n, Department of Physiology and Pharmacology, Karo- linska Institute, S-171 77 Stockholm, Sweden. (17, 18), but other investigators have shown no change at all (19, The present study was supported by the Swedish Medical Research Council (project 20) or altered function of the receptor (21). number 2553 to Bertil B. Fredholm and project number 5234 to Hugo Lagercrantz), the Institute for Scientific Information on Coffee, a Biomed II project funded by the European Caffeine given in doses similar to those resulting in long- Commission, The Swedish Society for Medical Research, and by Karolinska Institutet. term behavioral changes has been reported to alter the postnatal 177 178 ÅDE´ N ET AL. development of adenosine A1 receptors (22–24). However, N-methyl-flunitrazepam (flunitrazepam) (1 nM) (20). Nonspe- adaptive effects of low doses of caffeine have not been de- cific binding was determined using R-PIA (100 ␮M) (DPCPX- scribed. We have earlier shown that caffeine (0.3 g/L) in the binding) and 2-chloroadenosine (100 ␮M) (CGS 21680 drinking water given to the rat dam during gestation and binding), NECA (50 ␮M) (SCH 58261 binding), and diazepam lactation produces plasma concentrations in the pups of 0.4–2 (5 ␮M) (flunitrazepam binding). Ten-micrometer-thick sec- mg/L and that this treatment leads to reduced susceptibility to tions were preincubated in 170 mM Tris-HCl buffer containing hypoxic brain ischemia on postnatal d 7 (25), a stage when the 1 mM EDTA and 2 U/mL adenosine deaminase at 37° for 30 rat CNS maturity is thought to correspond to that of a near-term min. Sections were then washed twice for 10 min at 23° in 170 human fetus (for review see Ref. 26). mM Tris-HCl buffer. Incubations were performed for2hat23° The present study was designed to examine the influence of in 170 mM Tris-HCl buffer containing DPCPX (120 Ci/mmol, chronic pre- and postnatal treatment with a dose of caffeine 0.5 nM), CGS 21680 (42.1 Ci/mmol, 2 nM), or SCH 58261 similar to that used by humans on development of adenosine (0.2 nM) and 2 U/mL adenosine deaminase. In the experiments with DPCPX, 1 mM MgCl was added to preincubation and to A1 and A2A receptors and their corresponding mRNA as well 2 as on benzodiazepine binding sites representing in the rat pup incubation buffer. The incubation with DPCPX was done in the brain. presence of 100 ␮M guanosine triphosphate (GTP) to convert all the receptors to the low-affinity state for agonists and METHODS thereby remove all endogenous adenosine (28). Sections were then washed twice for 5 min each in ice-cold Tris-HCl, dipped Treatment. The experiments, which followed the European three times in ice-cold distilled water, and dried at 4° over a Community regulations, were approved by the regional animal strong fan. Slides were exposed to [3H]-sensitive film with [3H] ethics committee. Forty-four Wistar rats and their litters were microscales for 4–8 wk. GABAA receptor density was deter- used. Dams (n ϭ 23) were given caffeine in the drinking water mined using receptor autoradiography with flunitrazepam (1 (0.3 g/L), which was exchanged every third day to fresh nM). Nonspecific binding was determined using diazepam (5 solutions, from embryonic d (E) 2 throughout gestation and ␮M). Ten-micrometer-thick sections were preincubated in 170 postnatal life. Twenty-one dams received ordinary tap water. mM Tris-HCl buffer at 4° for 30 min. Incubation was per- The daily intake of water was measured in all litters. The day formed for1hat4°in170mMTris-HCl buffer containing when a vaginal plug was found was designated E0. Rat brains N-methyl-flunitrazepam (85 Ci/mmol, 1.0 nM). Sections were were examined at E14, E18, E21, exactly 2 h and 24 h after then washed twice for 1 min each in Tris-HCl at 4°, dipped in vaginal delivery (P2h, P24h), and at postnatal d 3 (P3), P7, P14, ice-cold distilled water, and dried at 4° over a strong fan. Slides and P21. From each treatment group 6 animals (from two different were exposed to [3H]-sensitive film with [3H] microscales for litters) were used for in situ hybridization and receptor binding 4–8 wk. studies. In situ hybridization. The 48-mer A1 adenosine receptor Plasma concentrations of caffeine. Trunk blood from fif- probe was complementary to nucleotides 985-1032 of the rat teen animals was collected in heparinized plastic tubes and A1 receptor (31). The 44-mer A2A probe was complementary to centrifuged. Plasma concentrations of caffeine and its metabolites nucleotides 916–959 of the dog RDC8 cDNA (32). The aden- theophylline, theobromine, and paraxanthine were analyzed using osine receptor probes have been tested for specificity (33, 34). high pressure liquid chromatography as described (27). The oligodeoxyribonucleotides were radiolabeled using termi- Sections. From embryos and pups up to P3, the whole head nal deoxyribonucleotidyl transferase and [35S] dATP to a was collected, whereas in older animals, the brain was rapidly specific activity of about 109 cpm/␮g. Slide-mounted sections dissected out. Heads and brains were frozen on dry ice and were hybridized in a cocktail containing 50% formamide, Ϫ ϫ ϫ stored at 80°C. Sagittal sections from the left hemisphere 4 SSC, 1 Denhardt’s solution, 1% sarcosyl, 0.02 M NaPO4 were cut on a Leitz cryostat. Sections were collected from the (pH 7.0), 10% dextran sulfate, 0.06 M dithiotreitol, 0.5 mg/mL lateral part of olfactory bulb toward midline.