The stimulatory action and the development of tolerance to caffeine is associated with alterations in gene expression in specific brain regions

Abstract

We sought neurochemical correlates to the stimulatory action of caffeine in rats and to adaptations during development of tolerance. Acute intraperitoneal injections of caffeine (7.5 mg/kg) increased locomotion and NGFI-A mRNA, a marker of neuronal activity, in the hippocampal area CA1, but decreased NGFI-A mRNA in rostral striatum and nucleus accumbens. Rats that received caffeine (0.3 gm/l) in their drinking water for 14 d developed tolerance to the stimulatory effect of a challenge with caffeine (7.5 mg/kg) and responded with a less pronounced decrease of NGFI-A mRNA in rostral striatum and nucleus accumbens. Metabolism of caffeine to its active metabolites was increased in tolerant animals, but the total level of active metabolites in brain was not significantly altered. Thus, there are changes in caffeine metabolism after long-term caffeine treatment, but they cannot explain development of tolerance. Caffeine-tolerant animals had downregulated levels of adenosine A2A receptors and the corresponding mRNA in rostral parts of striatum, but an increased expression of adenosine A1 receptor mRNA in the lateral amygdala. No changes in mesencephalic tyrosine hydroxylase mRNA were found in caffeine-tolerant rats. Thus, we have identified neuronal pathways that are regulated by adenosine A1 and/or A2A receptors and are targets for the stimulatory action of caffeine. Furthermore, adaptive changes in gene expression in these brain areas were associated with the development of locomotor tolerance to caffeine.

Fig. 1.

The development of tolerance to caffeine’s ability to increase locomotor activity over a course of chronic caffeine administration. Data are expressed as mean (±SEM) of total activity counts during a 60 min session. Asterisksindicate a significant difference between caffeine+saline- and caffeine+caffeine-treated animals. Plus signs indicate a significant difference between water+caffeine- and caffeine+caffeine-treated animals. ⁺ p< 0.05; **p < 0.01; ^+++,***p < 0.001.

Fig. 2.

Effects of saline or caffeine (7.5 mg/kg) challenge on locomotor activity (A), forward locomotion (B), rearing (C), locomotion in periphery (D), corner time (E), and locomotion in center (F) in animals treated chronically with water or caffeine (0.3 gm/l) for 14 d. Data are expressed as mean (±SEM) of total activity counts during a 60 min session. Asterisks indicate a significant difference between caffeine+saline- and caffeine+caffeine-treated animals.Plus signs indicate a significant difference between water+caffeine- and caffeine+caffeine-treated animals.⁺ p < 0.05; **p< 0.01; ^+++, ***p < 0.001.

Fig. 3.

Potency of caffeine, theophylline, paraxanthine, and theobromine as antagonists at striatal A₁ and A_2A receptors. A shows displacement of binding of 1 nm [³H]CHA from striatal A₁ receptors. Means (±SEM) from duplicate determinations from two separate experiments. B shows displacement of binding of 2 nm [³H]CGS 21680 from striatal A_2A receptors. Means (±SEM) from duplicate determinations from two separate experiments. Using theK_D value for CHA determined earlier (Johansson et al., 1993), the following K_ivalues for A₁ receptors were calculated: caffeine 20.2 μm, theophylline 4.7 μm, paraxanthine 5.0 μm, theobromine 98 μm. Using theK_D value for CGS 21680 determined earlier (Parkinson and Fredholm, 1990), the followingK_i values for A_2A receptors were calculated: caffeine 8.8 μm, theophylline 5.1 μm, paraxanthine 7.6 μm, theobromine 109 μm.

Fig. 4.

Quantitative measurements of gene expression and ligand binding were performed +3.20 mm (A), +1.20 mm (B), +0.48 mm (C), −0.92 mm (D), −3.14 mm (E), and −5.20 mm (F) from bregma. Gray squares delineate the regions examined.Amyg lat, Lateral amygdala; CA 1, field CA 1 of Ammon’s horn in hippocampus; CA 3, field CA 3 of Ammon’s horn in hippocampus; CC, cingulate cortex;CP, caudate-putamen; GD, gyrus dentatus;Gen, medial geniculate nucleus; GP, globus pallidus; MC, motor cortex; PFC, prefrontal cortex; Sep lat, lateral septum;SH, septohippocampal nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; SSC, somatosensory cortex;VTA, ventral tegmental area.

Fig. 5.

Color-coded photomicrographs showing binding of [³H] SCH58261 (A, B) and A_2A receptor mRNA (C, D), +1.20 mm from bregma, in water+saline-treated (A, C) and caffeine+saline-treated (B, D) animals. Eand F show A₁ receptor mRNA, −3.14 mm from bregma, in water+saline-treated (E) and caffeine+saline-treated (F) animals. Thewhite arrows point to the lateral amygdala.

Fig. 6.

Saturation curves of [³H]DPCPX binding in the CA 1 part of hippocampus (A) and of [³H]SCH 58261 binding in the lateral part of rostral striatum (B) from animals treated with water+saline, caffeine+saline, or caffeine(withdrawal)+saline. No significant differences in the B_max andK_D values for [³H]DPCPX-binding between the treatment groups were seen in substantia nigra pars reticulata, lateral geniculatum, or the hippocampal areas CA 1, CA 3, and gyrus dentatus. In contrast,B_max values were significantly lower in the rostrolateral (358 ± 10.3 fmol/gray matter) and rostromedial (341 ± 14.19 fmol/gray matter) parts of striatum in caffeine+saline- as compared with water+saline-treated animals (402 ± 7.13 and 382 ± 6.10 fmol/gray matter, respectively). No significant differences could be observed between water+saline-treated animals and caffeine(withdrawal)+saline-treated animals that showed B_max values of 380 ± 13.5 and 355 ± 16.4 fmol/gray matter in the rostrolateral and rostromedial parts of striatum. No significant changes inK_D values of [³H]SCH 58261 (average 0.20 nm) were seen between the different treatment groups in any of the areas investigated.

Fig. 7.

Histograms showing the effects of acute (A) or chronic (B) intraperitoneal administration of caffeine, at the indicated doses, on A_2A receptor mRNA. In C, the lack of effect of pretreatment with c-fos antisense oligonucleotide on reductions in A_2A receptor mRNA is shown.

Fig. 8.

Dark-field photomicrographs showing NGFI-A mRNA expression at +1.20 mm (A–D), +0.48 mm (E–H), −3.14 mm (I–L), and −5.20 mm (M–P) from bregma after water+saline (A, E, I, M), water+caffeine (B, F, J, N), caffeine+saline (C, G, K, O), and caffeine+caffeine (D, H, L, P).