Brain-Derived Neurotrophic Factor

Nunes et al. [57] examined the effects of caffeine (0.3 g/L) administered from childhood onwards in the BDNF and its related proteins in both sexes of SHR rats. BDNF and its related proteins were therefore evaluated in the hippocampus of WKYs and SHRs of both sexes at PND 55. A statistical analysis revealed a significant effect of strain on BDNF levels, while the precursor form (proBDNF) remained unaltered. The TrkB receptor full length (TrkB-FL), phospho-TrkB, and truncated-form TrkB receptors were immunodetected in the hippocampuses of the WKYs and SHRs of both sexes. A statistical analysis revealed a significant effect of strain on the truncated form and also on phospho-TrkB. Furthermore, the transcription factor CREB was not altered either by strain or sex, although its phosphorylated form (phospho-CREB) was increased in the SHR hippocampus from both sexes. Finally, Nunes et al. [57] evaluated the impact of caffeine only on the BDNF levels and TrkB receptors (TrkB-FL, phospho-TrkB, and TrkB-T). Caffeine administered from PND 15 up to PND 55 (caff/caff) reduced the BDNF levels in the hippocampuses of SHR male rats, whereas the BDNF levels were unaltered in the SHR female rats in both schedules of treatment. In the male rats, caffeine in both schedules of treatment did not change either

TrkB-FL or TrkB-T levels, whereas female SHRs showed reduced TrkB-FL and TrkB-T forms as a consequence of caffeine treatment. Neither the increased phospho-TrkB nor the CREB were modified in the hippocampuses of the SHRs following caffeine treatment.

#### Neuronal Development In Vitro

Alves et al. [63] investigated caffeine's in vitro effects at the neuronal level. At first, SHR and WKY rats' cultured frontal cortical neurons were immunostained for MAP-2 during in vitro development. Later on, somatodendritic analyses were performed, measuring branch point number, root number, and maximal and total neurite length. Neurons from the SHRs displayed fewer differentiation patterns, including neurite branching, shorter maximal neurite length, and decreased axonal outgrowth. Following a 24 h period of caffeine incubation (30 μM), the SHR neurons showed an inferior percentage of zero branch points, and a superior percentage of two branch points. A trend toward a superior percentage of one-branch-point-neurons was observed for SHR neurons following treatment with caffeine. Caffeine also promoted a rise in the total and maximal neurite length in neurons from both strains. PKA or PI3K inhibitor were subsequently used to study whether one of the transducing systems activated by adenosine receptors, and in the neuronal differentiation, are responsible for the effects produced by caffeine. PKA inhibitor KT5720 (5 μM) did not change caffeine's ability to augment the percentage of SHR neurons with more branch points. Caffeine's effect on the recovery of the total neurite length of the SHR neurons was obstructed by PKA inhibitor. Comparable results were seen for maximal neurite length, in which PKA inhibitor completely decreased caffeine's effects. Finally, LY294002 (50 μM) was used as an inhibitor of PI3K and its presence blocked caffeine's effect on the increase in the number of branch points in SHR neurons. Furthermore, caffeine's effect on the prevention of reductions in the total neurite length were eliminated in the presence of PI3K inhibitor. Similar results were found for the maximal neurite length. The number of roots was also reduced by PI3K inhibitor in SHR neurons.

#### **4. Discussion**

ADHD is characterized by symptoms including attention deficits, impulsivity, and hyperactivity [3,4] that frequently persist throughout life [1,2,6]. Prefrontal cortex function modulation and attentional/behavioral regulation depends on the optimal release of signalling molecules such as NE, DA [24,25], as well as 5-HT, GLU, or ACh [26–28]. In this respect, genes, including the DAT or the DRD4 [31,32] or the SERT, the SNAP-25, and the BDNF [29,30], might play a role in causing ADHD. Therefore, agents that can lead to the optimal balance of these organic compounds are hypothetically beneficial in patients with ADHD by mainly returning prefrontal activity to adequate functional levels [18,33]. In this sense, it has long been discussed whether caffeine could become an effective pharmacological compound for the management of symptoms of ADHD [64,65].

This systematic review analyzed 13 animal studies that investigated the effects of caffeine on the modulation of ADHD-like symptoms. Overall, the reviewed results show that caffeine treatment increases attention and improves learning, memory, and olfactory discrimination without altering blood pressure and body weight.

Regarding attention, caffeine treatment improved the attentional and behavioral flexibility of SHRs [51], the spatial attention of 6-OHDA lesioned rats [53], and SI in ICR mice [52] during adolescence. Caffeine treatment improved the reaction time of LE and CD rats [55] and focus and attention in zebrafish [54] during adulthood.

Regarding learning and memory, caffeine treatment plus physical exercise during adulthood and adolescence improved working memory in SHRs [56]. In the same vein, caffeine treatment alone restored non-associative learning in female SHRs [57], improved working memory in SHRs [59], female SHRs [62], and adolescent SHRs [57]. The administration of caffeine improved spatial learning deficit in SHRs, increased memory retention in WKY rats [60], and improved spatial short-term memory in SHRs [51] and female SHRs [57].

Concerning olfactory discrimination, caffeine treatment, together with physical exercise, was able to restore olfactory discrimination in SHRs during adolescence or adulthood [56]. Concerning blood pressure, caffeine treatment did not alter the hypertensive phenotype in SHR [60,62] during adolescence or adult life [56], nor during the adult female SHR prepubertal period [59]. Finally, caffeine treatment did not alter body weight in SHRs [51,59].

If we are ever to acquire a truly in-depth understanding of ADHD pharmacotherapy, we need to face the following question: Does caffeine deserve a place in the battery of pharmacological agents for ADHD treatment, particularly during adolescence? Although previous meta-analyses [64] and reviews [65] were unable to provide any recommendations for adolescents diagnosed with ADHD, due to a lack of data, our reviewed results provide updated preclinical evidence and support the therapeutic potential of caffeine to improve attention, learning, memory, or olfactory discrimination in ADHD, especially during adolescence.

Beyond its clear effects on improving performance in tasks requiring attention, learning, memory and olfactory discrimination, without altering blood pressure and body weight, the implication of caffeine in modulating ADHD-like hyperactivity symptoms remains controversial. Indeed, caffeine treatment plus physical exercise did not affect locomotor activity in SHRs [56]. In a similar manner, caffeine treatment alone did not alter locomotion in SHR [51,59,60], preadolescent SHR [57], or young LDLr mice [58]. Nonetheless, caffeine treatment did increase locomotor activity in adolescent female SHRs [57], zebrafish [54]. Furthermore, it produced an increase related to dose in locomotion in CD rats and a significant attenuation of CGS-21680-induced hypolocomotion in CD rats [55], and it attenuated locomotor activity in middle-aged LDLr mice [58] and 6-OHDA lesioned rats throughout the prepubertal period [53]. This apparent discrepancy may have resulted from caffeine 's promotion of different effects according to age and sex. In this regard, Nunes et al. [57] suggested that the intake of caffeine from the childhood period onwards may aggravate hyperactivity in females, if the consumption continues up to the adolescence period. Szczepanik et al. [58] linked the age-dependent effect induced by caffeine with the idea that the blockade of adenosine A1/A2A receptors attempts to renormalize a potentially maladaptive system [66], with age an important escalating factor in mice. In a different study, Ruiz-Oliveira et al. [54] proposed that caffeine-induced bursts of locomotion may be caused by a decrease in fatigue [67] rather than by an anxiogenic response. Importantly, the attenuation of motor activity by caffeine consumption was determined as a natural effect of growth rather than an effect of caffeine intake by Caballero et al. [53].

In terms of impulsivity, although acute pretreatment with caffeine increased the number of large-reward choices made by SHRs, chronic treatment with caffeine increased the impulsive phenotype and decreased choices of large rewards by SHRs [61]. This discrepancy may be explained by previous studies performed on animal models of brain diseases, showing that while acute treatment acts mainly on A1 receptors, chronic treatment acts mainly on A2A receptors [68]. Leffa et al. [61], in this direction, underscored the ability of the adenosine modulation system to control behavioral inhibition.

Besides reviewing animal studies deciphering the effects of caffeine in the modulation of ADHD-like symptoms, we reviewed for the first time animal studies examining the effects of caffeine and adenosine receptors on neurons isolated from SHRs, at the neuronal level.

In this respect, treatment with caffeine and physical exercise during the adolescence period augmented the quantity of SNAP-25, syntaxin, and serotonin in the prefrontal cortex and the hippocampus, as well as striatal dopamine quantity, in SHRs [56]. In a similar manner, caffeine treatment alone during the adolescence period attenuated the improvement in DAT density in the fronto-cortical and striatal terminals of SHRs and diminished the dopamine uptake by synaptosomes from SHRs' fronto-cortical and striatal terminals [51]. Furthermore, Pandolfo et al. [51] demonstrated that fronto-cortical nerve terminals are provided with AdenosineA2A receptor, the target of chronic caffeine exposure, whose density was found to be increased in SHRs. Caffeine treatment normalized BDNF levels in the hippocampuses of SHR males, while the same treatment normalized TrkB receptors TrkB-FL and TrkB-T SHR in the hippocampuses of SHR females [57]. Finally, neurons from SHRs showed an inferior number of zero-branch points, and a superior number of two-branch-points-neurons following in vitro caffeine treatment consisting of 24 h of caffeine incubation. After treatment with caffeine, an increase in the total and maximal neurite length and a tendency toward a superior number of one-branch-point neurons was also observed for SHR neurons. The effect of caffeine on increasing maximal neurite length, and on recuperating the entire neurite length of neurons from SHR, was entirely blocked by PKA inhibitor. LY294002, as an inhibitor of PI3K, blocked caffeine's effects on the increase in the amount of branch points in SHR neurons. Finally, the effect of caffeine on the prevention of reductions in the total neurite length, increasing maximal neurite length, and the number of roots was eradicated by the presence of PI3K inhibitor in SHR neurons [63].
