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. 2019 Apr 20;11(4):888.
doi: 10.3390/nu11040888.

Theobromine Improves Working Memory by Activating the CaMKII/CREB/BDNF Pathway in Rats

Rafiad Islam  1   2 , Kentaro Matsuzaki  3 , Eri Sumiyoshi  4 , Md Emon Hossain  5   6 , Michio Hashimoto  7 , Masanori Katakura  8   9 , Naotoshi Sugimoto  10   11 , Osamu Shido  12
Affiliations

Theobromine Improves Working Memory by Activating the CaMKII/CREB/BDNF Pathway in Rats

Rafiad Islam et al. Nutrients. .

Abstract

Theobromine (TB) is a primary methylxanthine found in cacao beans. cAMP-response element-binding protein (CREB) is a transcription factor, which is involved in different brain processes that bring about cellular changes in response to discrete sets of instructions, including the induction of brain-derived neurotropic factor (BDNF). Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been strongly implicated in the memory formation of different species as a key regulator of gene expression. Here we investigated whether TB acts on the CaMKII/CREB/BDNF pathway in a way that might improve the cognitive and learning function in rats. Male Wistar rats (5 weeks old) were divided into two groups. For 73 days, the control rats (CN rats) were fed a normal diet, while the TB-fed rats (TB rats) received the same food, but with a 0.05% TB supplement. To assess the effects of TB on cognitive and learning ability in rats: The radial arm maze task, novel object recognition test, and Y-maze test were used. Then, the brain was removed and the medial prefrontal cortex (mPFC) was isolated for Western Blot, real-time PCR and enzyme-linked immunosorbent assay. Phosphorylated CaMKII (p-CaMKII), phosphorylated CREB (p-CREB), and BDNF level in the mPFC were measured. In all the behavior tests, working memory seemed to be improved by TB ingestion. In addition, p-CaMKII and p-CREB levels were significantly elevated in the mPFC of TB rats in comparison to those of CN rats. We also found that cortical BDNF protein and mRNA levels in TB rats were significantly greater than those in CN rats. These results suggest that orally supplemented TB upregulates the CaMKII/CREB/BDNF pathway in the mPFC, which may then improve working memory in rats.

Keywords: BDNF; CREB; CaMKII; behavior; cacao; theobromine; working memory.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schedule for Experiment 1 (radial arm maze (RAM) task).
Figure 2
Figure 2
Schedule for Experiment 2 (Y-maze test and novel object recognition (NOR) test).
Figure 3
Figure 3
The effect of theobromine (TB) on (A) working memory errors (WME); (B) reference memory errors (RME); and (C) latency assessed by the radial arm maze task. Results are expressed as the mean ± S.E.M. (n = 12 for each group). *** p < 0.001; significant difference between groups.
Figure 4
Figure 4
The effect of theobromine (TB) on the brain-derived neurotrophic factor (BDNF) protein level in the medial prefrontal cortex (mPFC). BDNF protein level in the mPFC of TB-fed rats (TB rats) was significantly higher than that of control rats (CN rats). Hippocampal BDNF protein levels did not differ between groups (Figure S1). Values are the mean ± S.E.M. (n = 5 for each group). ** p < 0.01; significant difference between groups.
Figure 5
Figure 5
The effect of theobromine (TB) on locomotor activity and spontaneous alteration behavior in rats assessed by the Y-maze test. (A) Locomotor activity of TB-fed rats (TB rats) and control rats (CN rats). There were no significant differences between groups in both trials. (B) TB rats had a significantly higher number of alteration in both trials compared to CN rats. Values are the mean ± S.E.M. (n = 8 for each group). * p < 0.05, ** p < 0.01; significant difference between groups.
Figure 6
Figure 6
The effect of theobromine (TB) on memory performance assessed by the novel object recognition (NOR) test. The discrimination index (DI) for exploring the object A1, A2 (A); object A1, B (B); and object A1, C (C). Both DIs (Object A1, B and Object A1, C) were significantly greater in TB-fed rats (TB rats) in 2 trials. Values are the mean ± S.E.M. (n = 5 for each group). * p < 0.05, ** p < 0.01; significant difference between groups. Total exploration time for same object (object A1, A2) (D); total exploration time for each object (object A1, B) (E); and total exploration time for each object (object A1, C) (F). TB rats stayed significantly longer time at the new object B and C in 2 trials. Values are the mean ± S.E.M. (n = 5 for each group). * p < 0.05, ** p < 0.01; significant difference between A1 and B or C.
Figure 7
Figure 7
Phospho-CaMKII (p-CaMKII) and phospho-CREB (p-CREB) levels in the medial prefrontal cortex (mPFC) of control rats (CN rats) and theobromine (TB)-fed rats (TB rats). TB enhanced (A) p-CaMKII; and (B) p-CREB levels in the mPFC. Values are the mean ± S.E.M. (n = 6 per group). * p < 0.05, ** p < 0.01; significant difference between groups.
Figure 8
Figure 8
The effect of theobromine (TB) on brain derived neurotrophic factor (BDNF) mRNA and protein levels. (A) Relative mRNA expression and (B) protein level in TB-fed rat (TB rats) of BDNF were significantly higher than those of control rats (CN rats) in the prefrontal cortex. Hippocampal BDNF protein levels did not differ between groups (Figure S2). Values are the mean ± S.E.M. (n = 6 for each group). * p < 0.05; significant difference between groups.
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