Changes in adenosine A1 receptor-mediated regulation of hippocampal area CA2 following chronic high-dose caffeine treatment during adolescence in rats

Memory is a highly complex cognitive function that involves interactions between numerous brain structures, proteins and signalling molecules. Indeed, experience-dependent changes in the strength of synaptic connections between neurons are though to underlie information storage in the brain. Thus, understanding the highly elastic nature, or the 'plasticity', of synapses is critical to uncovering how the brain converts our individual experiences into lasting memories. To this end, modulatory neurotransmitter systems play a central role in shaping synaptic processes involved in the encoding and retention of information within neural circuits. A fundamental problem remains, though, in understanding how extrinsic factors recruit, mimic or even hijack these endogenous regulatory signals to affect the development of brain systems critical to cognitive and mnemonic function.

The mammalian nervous system undergoes several critical periods during early postnatal development in which cognitive and sensory functions are sensitive to lasting disruption by an array of environmental factors. Indeed, there is growing evidence to suggest that the hippocampal purinergic system in rodents also undergoes a period of significant maturation related to the expression pattern of the adenosine A1 receptor (A1R). Adenosine acts as a neuromodulator in the brain, and signalling at adenosine receptors in the hippocampus has been shown to influence memory formation and behaviour by regulating synaptic plasticity. In rats, the expression pattern of the A1R is not static during early development. In particular, the surface expression of the A1R increases significantly during adolescence in a specific part of the hippocampus, known as area Cornu Ammonis 2 (or CA2). However, changes in synaptic function and neuronal physiology in area CA2 following manipulations to disrupt adenosinergic signalling during development have yet to be explored in animal models.

Consistent with the high expression of A1Rs in CA2 is the observation that caffeine and other A1R blockers preferentially enhance excitatory synaptic transmission in area CA2 at concentrations that have little effect on responses in other parts of the hippocampus. This suggests that A1Rs in CA2 may play a central role in mediating the cognitive enhancing effects of caffeine. Caffeine is a naturally-occurring cognitive enhancer that is widely-consumed to improve attention and augment memory. Its primary mechanism of action is thought to be through blockade of A1Rs, however, little is known about hippocampal area CA2 where A1Rs are highly expressed and where the surface expression of the A1R changes dramatically during adolescent development.

The popularity of highly-caffeinated so-called 'energy' drinks has increased dramatically in the past 15 years, and recent trends highlight how teenagers consume energy drinks regularly and in quantities that exceed recommended daily limits. It is currently unknown, though, whether habitual consumption of energy drinks during adolescence is associated with lasting changes in brain function. Given that A1Rs increase dramatically during adolescence, together with the specificity that caffeine has in modifying synaptic function in CA2, the aim of this proposal is to evaluate the long-term effects of chronic caffeine administration on CA2 physiology and CA2-dependent forms of cognition. To achieve this, human adolescent energy drink consumption patterns will be modelled experimentally in rats and caffeine-induced changes in synaptic function, neuronal morphology and protein expression in CA2 will be assessed at the end of the dosing regimen. Evidence in support of a developmental window during which adolescents may be susceptible to lasting neurological dysfunction resulting from habitual caffeine consumption may provide justification to amend policy and regulate the sale of highly caffeinated products to vulnerable populations.

Caffeine enhances cognition, in part, by blocking adenosine receptors in the hippocampus. Specifically, bidirectional changes in synaptic efficacy mediated by A1Rs in CA2 may contribute significantly to the cognitive enhancing effects of caffeine. Interestingly, A1R expression increases markedly in CA2 during adolescence, suggestive of a critical period in the development of the hippocampal purinergic system. The aim of this proposal is to determine whether chronic exposure to high levels of caffeine during adolescence is sufficient to disrupt hippocampal function later in adulthood. This will be achieved by dosing rats daily with caffeine from P21 to P70 in order to mimic human adolescent caffeine consumption patterns. Changes in A1R-mediated regulation of synaptic function, neuronal morphology and protein expression, as well as mechanisms central to CA2-dependent forms of cognition, including social recognition memory will be assessed at the end of the dosing regimen.

Multiple techniques will be employed. Behavioural assays and intracellular recordings of synaptic and intrinsic conductances in CA2 neurons will be used to determine whether chronic caffeine influences CA2-dependent forms of cognition by altering long-term neuronal excitability and synaptic function following tonic blockade of A1Rs. Next, Golgi staining and confocal imaging of neurons and spines, respectively, will be used to assess caffeine-induced changes in neuronal morphology in CA2. Immunolabelling for proteins, including markers for A1Rs, glial cells and interneurons will be used to assess caffeine-induced changes in protein expression and network development in the hippocampus. Finally, intracellular recordings of synaptic responses evoked by alternating stimulation of converging inputs to CA2 in naive brain slices exposed to adenosine receptor-selective compounds will be used to determine whether A1R-dependent forms of synaptic plasticity differ in separate anatomical inputs to area CA2.

The contributions of hippocampal area CA2 to cognitive and mnemonic function are largely unknown, but recent evidence indicates that CA2 may play an important role in mediating social memory and aggression. The work covered in this proposal is designed to assess whether the increase in A1R expression observed in area CA2 during adolescent development is susceptible to disruption by chronic high-dose caffeine treatment. Experiments are designed to mimic recent patterns observed in teenage youths in which highly-caffeinated beverages are consumed regularly and in significant quantities. Although caffeine is generally considered safe when consumed in moderation, it is unknown whether habitual consumption of high quantities of caffeine, such as those found in some energy drinks, is causing significant and lasting adaptations in brain function, particularly in hippocampal area CA2.

In line with the stated goals, adolescent consumers are perhaps the largest benefactors of the proposed work. Indeed, since it is primarily teenagers and young adults who are consuming energy drinks and other related products habitually, these individuals are likely the ones who are the most at risk from any potentially deleterious effects associated with chronic high-dose caffeine use. In particular, lasting impairments in social behaviour and social memory may be a direct consequence of caffeine-induced changes in CA2 function mediated by tonic inhibition of A1Rs. Results from experiments described in this proposal could help inform healthy lifestyle choices when teenagers are faced with decisions related to their drinking habits and beverage options. As such, adolescent consumers should be able to shield themselves from potentially irreversible damage to a brain structure that has only recently been shown to mediate an array of social behaviours.

Other stakeholders who would benefit directly from the work described in this proposal include government and industry policy makers, as well as local charities with a focus on adolescent health issues. Data in support of lasting impairments in cognitive function resulting from habitual high-dose caffeine use in adolescent youths would provide government representatives and industry stakeholders with suitable justification to implement new policies designed to regulate the sale and marketing of highly-caffeinated beverages to vulnerable populations. An increase in scientifically-informed awareness regarding potential risk factors associated with habitual high-dose caffeine use in teenagers and young adults may also help attract additional funding and investment from youth charities to foster continued research into A1R-mediated regulation of CA2 function.

The general public is also likely to benefit from increased awareness of potential dangers associated with habitual high-dose caffeine use on brain development and cognition. Parents will be able to make make informed decisions regarding whether they should purchase products containing excessive amounts of caffeine for their children and educators can work with adolescents directly to foster healthy habits and smart beverage choices. The ability to sway public opinion is a powerful means through which change can be introduced, and increased public awareness regarding putative dangers associated with habitual high-dose caffeine use may embolden the general public to increase pressure on government and industry to enact further legislation.

The pharmaceutical industry may also benefit directly from the proposed research. Given the role of hippocampal area CA2 in mediating social behaviours together with the fact that mice lacking A1Rs tend to be more anxious and aggressive, signalling pathways linked to A1Rs in area CA2 may represent novel therapeutic targets in the treatment of social disorders, including anxiety.