Aged mice receiving caffeine since adulthood show distinct patterns of anxiety-related behavior

Highlights

• Anxiety behavior displayed by aged mice was test dependent manner.

• Caffeine did not affect locomotion and anxiety behavior in the open field.

• Moderate caffeine prevents changes in anxiety with aging in the elevated plus maze.

• Moderate caffeine prevents age-related increase in cortical SNAP-25.

• Caffeine did not modify the age-related increase in cortical adenosine A2A receptors.

Abstract

Caffeine is the psychostimulant most consumed worldwide. Anxiogenic effects of caffeine have been described in adult animals with controversial findings about its anxiogenic potential. Besides, the effects of caffeine on anxiety with aging are still poorly known. In this study, adult mice (6 months old) started to receive caffeine (0.3 and 1.0 mg/mL, drinking water) during 12–14 months only in the light cycle and at weekdays. The open field (OF) and elevated plus maze (EPM) testing were used to determine the effects of caffeine on anxiety-related behavior in adult and aged mice (18–20 months old). Because aging alters synaptic proteins, we also evaluated SNAP-25 (as a nerve terminals marker), GFAP (as an astrocyte marker) and adenosine A₁ and A_2A receptors levels in the cortex. According to the OF analysis, caffeine did not change both hypolocomotion and anxiety with aging. However, aged mice showed less anxiety behavior in the EPM, but after receiving caffeine (0.3 mg/mL) during adulthood they were anxious as adult mice. While SNAP-25 and adenosine A_2A receptors increased with aging, both GFAP and adenosine A₁ receptors were not affected. Caffeine at moderate dose prevented the age-related increase of the SNAP-25, with no effect on adenosine A_2A receptors. The absence of effect for the highest dose suggests that tolerance to caffeine may have developed over time. Aged mice showed high responsiveness to the OF, being difficult to achieve any effect of caffeine. On the other hand this substance sustained the adult anxious behavior over time in a less stressful paradigm, and this effect was coincident with changes in the SNAP-25, suggesting the involvement of this synaptic protein in the ability of caffeine to preserve changes related to emotionality with aging.

Introduction

Caffeine is the major psychostimulant consumed worldwide, which at doses regularly consumed is a non-selective antagonist of adenosine A₁ (A₁R) and A_2A (A_2AR) receptors [1]. Over the last years, experimental studies have reported preventive effects of caffeine against memory impairment with aging [2], [3], [4] and in rodent models of Alzheimer's disease [5], [6], [7], [8], [9]. Epidemiologic evidences have also supported that caffeine consumption prevents cognitive decline with aging and the risk for developing Alzheimer's disease [10], [11]. While these benefits promoted by chronic caffeine intake have been well documented, the effects of caffeine on anxiety-related behavior are more controversial.

In human subjects, caffeine has been reported to augment anxiety levels when consumed at moderate and high doses [12], [13]. In rodents, caffeine acutely administered at high doses increases anxiety [14], [15], [16], [17]. On the other hand, anxiolytic effects have been found in acute administrations with similar doses aforementioned, suggesting that anxiolytic/anxiogenic effects of caffeine are not necessarily dose-related [18], [19]. Chronic consumption can also be anxiogenic [16], though tolerance to caffeine can develop over time [14]. The reason for such distinct findings is resulting from differences between tests employed to assess anxiety, strains and sex [20]. More recently, some reports have found an association between caffeine use and anxiety in the adolescent population [21], and adolescent animals being more responsive to anxiogenic effects of the caffeine [22], [23], [24], [25]. However, most studies on the effects of caffeine on anxiety are conducted in adult animals [16], [17], [18], [19], [26], [27].

While the impact of caffeine exposure on memory has received considerable attention, little is known about its effects on the emotionality with age. Anxiety disorders are highly prevalent across the lifespan [28], and therefore cases of anxiety in the elderly are commonly assumed to represent the continuing chronic course of early onset illness and/or a severity marker of depression [29]. In this scenario, chronic caffeine was able to prevent memory impairment, but not anxiety in a depression-prone mouse strain [30]. On the other hand, caffeine blunted anxiety triggered by chronic unpredictable stress [27] and social defeat stress [31]. Therefore, many studies on effects of caffeine were focused in the prevention of age-related memory impairment, while its impact in the anxiety with aging has received little attention.

Given that anxiety disorders are highly prevalent across the lifespan [28], the effects of a long-term caffeine treatment were investigated in two paradigms widely used to assess anxiety-related behavior. Besides, synaptic proteins usually change with aging. Therefore, we also examined an association of the impact of long term caffeine on anxiety with synaptic proteins, which were assessed in the cortex, such as SNAP-25 (as a nerve terminals marker), GFAP (as an astrocytic marker) and A₁R and A_2AR the main targets of caffeine.