Adolescent caffeine consumption increases adulthood anxiety-related behavior and modifies neuroendocrine signaling

Abstract

Caffeine is a commonly used psychoactive substance and consumption by children and adolescents continues to rise. Here, we examine the lasting effects of adolescent caffeine consumption on anxiety-related behaviors and several neuroendocrine measures in adulthood. Adolescent male Sprague-Dawley rats consumed caffeine (0.3g/L) for 28 consecutive days from postnatal day 28 (P28) to P55. Age-matched control rats consumed water. Behavioral testing for anxiety-related behavior began in adulthood (P62) 7 days after removal of caffeine. Adolescent caffeine consumption enhanced anxiety-related behavior in an open field, social interaction test, and elevated plus maze. Similar caffeine consumption in adult rats did not alter anxiety-related behavior after caffeine removal. Characterization of neuroendocrine measures was next assessed to determine whether the changes in anxiety were associated with modifications in the HPA axis. Blood plasma levels of corticosterone (CORT) were assessed throughout the caffeine consumption procedure in adolescent rats. Adolescent caffeine consumption elevated plasma CORT 24h after initiation of caffeine consumption that normalized over the course of the 28-day consumption procedure. CORT levels were also elevated 24h after caffeine removal and remained elevated for 7 days. Despite elevated basal CORT in adult rats that consumed caffeine during adolescence, the adrenocorticotropic hormone (ACTH) and CORT response to placement on an elevated pedestal (a mild stressor) was significantly blunted. Lastly, we assessed changes in basal and stress-induced c-fos and corticotropin-releasing factor (Crf) mRNA expression in brain tissue collected at 7 days withdrawal from adolescent caffeine. Adolescent caffeine consumption increased basal c-fos mRNA in the paraventricular nucleus of the hypothalamus. Adolescent caffeine consumption had no other effects on the basal or stress-induced c-fos mRNA changes. Caffeine consumption during adolescence increased basal Crf mRNA in the central nucleus of the amygdala, but no additional effects of stress or caffeine consumption were observed in other brain regions. Together these findings suggest that adolescent caffeine consumption may increase vulnerability to psychiatric disorders including anxiety-related disorders, and this vulnerability may result from dysregulation of the neuroendocrine stress response system.

Keywords: Adolescence; Corticotropin-releasing hormone; Glucocorticoid; Hypothalamic–pituitary–adrenal axis; Immediate early gene.

Figure 1

Adolescent caffeine consumption does not produce significant alterations in fluid consumption or body weight outcomes over the course of the caffeine consumption procedure. A) Male Sprague-Dawley rats received caffeine or water for 28 days beginning on P28. Behavioral testing, neuroendocrine measures, and tissue collection occurred after 7 days in the absence of caffeine. B) Caffeine-consuming adolescent rats gained weight equivalently to control rats that had ad libitum access to water. C) The volume of fluid consumed by the two groups was equivalent throughout the procedure. D) The amount (mg/kg) of caffeine consumed was assessed throughout the procedure and resulted in a progressive decrease in caffeine intake over the course of the procedure. n = 15/group

Figure 2

Adolescent caffeine consumption, but not adult caffeine consumption, increases anxiety-related behaviors. A) Rats that consumed caffeine during adolescence spend less time on the open arms (left) and enter the open arms less frequently (right) when exposed to the elevated plus maze. n = 9-10/group. B) Rats exposed to caffeine during adulthood show no differences in time spent on or entries into the open arms of the elevated plus maze. n = 8/group. C) Caffeine consumption during adolescence decreases time spent in the center of an open field chamber (left), but does not change overall locomotion (right). n = 4/group. D) Caffeine consumption during adulthood has no effect on open field behavior (left) or total locomotion (right). n = 8/group. E) Chronic caffeine consumption during adolescence decreases social interaction in adulthood. n = 8-10/group. F) Chronic caffeine consumption during adulthood has no effect on social interaction. n = 8/group. E) * significant from water controls (p< 0.05)

Figure 3

Increased anxiety-related behavior was observed in both adolescent and adult animals during the last week of caffeine consumption. A) Rats consuming caffeine during adolescence spend less time on the open arms (left) when tested on the elevated plus maze during the last week of caffeine consumption. n = 20/group. B) Rats consuming caffeine during adulthood also spend less time on the open arms (left) and enter the open arms less frequently (right) when tested on the elevated plus maze during the last week of caffeine consumption. n = 9-10/group. * significant from water controls (p< 0.05)

Figure 4

Adolescent caffeine consumption produces HPA axis dysregulation. A) Basal plasma corticosterone (CORT) levels across the course of the caffeine consumption paradigm. Caffeine consumption initially increases basal CORT, but rats appear to become tolerant to the HPA-axis activating effects of caffeine by day 14. Twenty-four h after the removal of caffeine rats show increased basal CORT. n = 8-10/group B) Basal CORT measures at the circadian trough and peak following a 7 day washout period without caffeine. Rats that consumed caffeine exhibit persistent CORT elevations at the circadian trough, but no differences in peak CORT levels. n = 7-8 C) Plasma adrenocorticotropin hormone (ACTH) levels were decreased in rats that consumed caffeine during adolescence following pedestal stress (t₂₆ = 2.474, p< 0.05, n = 7-8). D) Plasma CORT levels were also decreased in adolescent caffeine consuming rats following pedestal stress (t₂₆ = 4.323, p< 0.001, n = 7-8). E) A peripheral injection of ACTH (1 mg/kg) induced significantly higher levels of plasma CORT in water control rats compared to rats that consumed caffeine during adolescence. n = 8-10/group * significant from respective water control group (p< 0.05), ‡ significant from Day 1 (p< 0.05), ^ significant from respective No Stress condition (p< 0.05), # significant (p< 0.001) from plasma CORT levels at the circadian trough

Figure 5

Chronic caffeine consumption during adolescence alters basal c-fos mRNA expression and c-fos mRNA expression in response to a psychological stressor. A) Stress increases c-fos mRNA expression in the PVN. Caffeine consumption during adolescence enhanced basal expression of c-fos mRNA in the PVN, but did not alter stress-induced c-fos expression. n = 6-9/group B) Pedestal stress increases in c-fos mRNA expression in the central amygdala (CeA) of control rats, but not rats that consumed caffeine during adolescence. n = 5-9/group. C) Pedestal stress increases c-fos mRNA in the basolateral amygdala (BLA) in both water and caffeine consuming groups. No Stress: n = 6-9/group. D) Pedestal stress has no effect on c-fos mRNA in the bed nucleus of the stria terminalis anteroventral portion (BSTav). No Stress: n = 5-9/group. E) Pedestal stress increases c-fos mRNA in the PFC in both water and caffeine consuming groups. No Stress: n = 6-9/group. F) Both adolescent caffeine and water groups show a stress-induced increase in c-fos mRNA expression in the nucleus accumbens shell (NAc shell). No Stress: n = 6-9/group. * significant from respective water control group (p< 0.05), # significant main effect of stress (p< 0.05)

Figure 6

Adolescent caffeine consumption produces increases Crf mRNA expression in the CeA. A) Both caffeine and water groups show the same levels of Crf mRNA in the PVN. n = 9-16/group. B) Chronic caffeine consumption increases Crf mRNA expression in the CeA No Stress: n = 5-8/group. C) There is no effect of adolescent caffeine consumption on Crf mRNA expression in the PFC. n = 6-9/group. D) Crf mRNA expression in the BSTav is not altered by caffeine consumption during adolescence. * significant from water controls (p< 0.05)