Repeated unpredictable stress blunts alcohol-induced memory deficit in adolescent rat

Abstract

Objective:

There exists a complex interaction between alcohol and stress on brain and behavior. Alcohol and stress are both known to affect memory. Whether stress and alcohol together can modulate memory functions in adolescent rats is not known. In the present study, effects of repeated unpredictable stress (RUPS) on contextual fear conditioning, a hippocampus-related memory function, were investigated in alcohol-treated adolescent rats.

Methods:

Rats were divided into four experimental groups: group i. saline-treated non-stressed rats (sal no stress), group ii. alcohol-treated non-stressed rats (alc no stress), group iii. saline-treated rats subjected to stress (sal + RUPS), group iv. alcohol-treated rats subjected to stress (alc + RUPS). All rats were trained in the fear conditioning paradigm, and 24 h later were tested for contextual fear conditioning in the conditioning chamber, and nonspecific fear memory in a modified chamber.

Results:

Stress, in the presence or absence of alcohol, did not alter nonspecific fear. RUPS exposure did not affect contextual freezing in vehicle-treated adolescent rats. Compared to vehicle-treated non-stressed rats, alcohol-treated non-stressed rats showed significant impairments in contextual freezing. Alcohol-treated RUPS rats performed better in the contextual freezing task than alcohol-treated non-stressed rats.

Conclusions:

RUPS exposure did not alter contextual fear conditioning in adolescent rats. Alcohol significantly reduced contextual fear memory in non-stressed rats. Alcohol-treated RUPS rats showed significantly better memory than alcohol-treated non-stressed rats. Together, these data suggest resiliency to stress-induced memory impairment in adolescent rats, and RUPS exposure causes blunting of alcohol’s negative effects on contextual fear conditioning.

Introduction

Adolescence is characterized by changes in brain structure and function [1]. The hippocampus undergoes significant maturation during the adolescent period, and it is associated with alterations in neurobiological and cognitive functioning [2]. Several major neuropsychiatric disorders such as anxiety, depression, schizophrenia and addiction, make their first appearance during adolescence [3].

Hippocampus, an important neural substrate for memory, is vulnerable to stress. Stress has been shown to impair hippocampus-associated memory functions [4]. Hippocampal slices prepared from rats exposed to stress show impairments in long term potentiation, a form of synaptic plasticity underlying learning and memory [5]. The adolescent brain is particularly sensitive to stress [6].

Adolescents differ from adults in their sensitivity to alcohol’s neurobehavioral effects. Our laboratory and others have shown that alcohol causes significant impairments in memory functions in adolescent rats [7,8].

Alcohol and stress are known to impair memory formation. Since the hippocampus is sensitive to both alcohol and stress, we hypothesized that adolescent rats subjected to both stress and alcohol would show greater impairments in hippocampus-related memory formation, than when exposed to either alone. In the present study, adolescent female rats were exposed to repeated unpredictable stress [RUPS], consisting of random intermittent exposures to a variety of stressors, in the presence or absence of alcohol treatment, and tested for contextual fear conditioning, a hippocampus-related memory task.

Materials and Methods

Subjects:

Forty-six female adolescent (PD25 at the beginning of experiment) Sprague Dawley rats (Taconic, Germantown, NY, USA) were used. Rats were housed in the temperature- and humidity-controlled institutional animal facility that was maintained on a 12h/12h light-dark cycle. Rats had ad libitum access to food and water, except for rats that were subjected to temporary food deprivation. All experimental protocols were approved by the Institution’s Animal Care and Use Committee and in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Alcohol treatment:

Alcohol was administered intraperitoneally (IP) at a dose of 2g/kg; the dose was selected based upon previous studies from our lab [8, 9]. Control rats received equivalent volumes of saline.

Unpredictable stressors:

Several stressors were used (Table 1 provides sequence of stressors applied).

Table 1.

Repeated unpredictable stress regime

Time	Day 1	Day 2	Day 3	Day 4
morning	NS	cage tilt 1 h	wet bedding 1h	cat urine 1 h
afternoon	cold swim 5 min	cat urine 1 h	restraint 1 h	NS
overnight	food deprivation	sleep deprivation	NS	social isolation + light on

NS = no stress exposure

Cold water swim stress:

A mixture of ice cubes and tap water was added to a plastic cage; temperature of the ice-water mixture was maintained at 4°C. Rats were placed individually in the ice-water mixture for 5 min. Control rats were placed in 25°C water for 5 min.

Overnight food deprivation stress:

Rats did not have access to food at night for 12 hours.

Cage tilt stress:

Rats were placed in individual cages that were kept inclined at 30 degrees for one hour.

Cat urine stress:

Rats were individually placed in cages that contained an open jar of bobcat urine, for one hour. Cages with bobcat urine were kept in a biological hood to prevent other rats from being inadvertently exposed to bobcat urine.

Sleep deprivation stress (overnight interrupted sleep):

Each animal was placed overnight on an upside-down small beaker kept in a plastic cage containing room temperature water. Every time the rat went into deep sleep and lost motor control, it would fall into the water. This would wake up the rat and it would climb back onto the beaker. Control rats were placed on an upside-down large beaker that had enough space for the rat to curl up comfortably and not fall off during deep sleep.

Wet bedding stress:

Water was added to cage bedding to make it soaking wet. Rats were kept individually in wet cages for one hour.

Restrain stress:

Rat was physically restrained in a restraining bag for one hour.

Overnight social isolation and light stimulation:

Rats were singly housed overnight, and room lights were kept on all night.

Behavioral testing

Fear conditioning: Protocol used for contextual fear conditioning was as described previously [10]. Briefly, rats were placed in a rat conditioning chamber (Coulbourne Instruments, PA). Acclimation: One day before training each rat was placed in the conditioning chamber for 15 min. Training: Rats were subjected to two cycles of conditioned stimulus (CS)-unconditioned stimulus (US) pairing. After the rat was introduced in the fear conditioning chamber, a sound stimulus (2.9 kHz) was played for 30 sec, and during the last two seconds a foot shock (2mA) was applied. Following a 90 sec inter-trial interval, the rat was subjected to a second trial; the second trial was identical to the first trial. Testing for fear: Twenty-four hours after training, each rat was placed in a modified chamber. The conditioning chamber was modified by partitioning it diagonally with a piece of plastic (change shape of the chamber from a square one to a triangular one), a sheet of smooth plastic was placed on the floor (conceal the metal grid floor underneath), and walls were covered with paper that had black vertical stripes painted on it (visual alteration). Rat was placed on one side of the divider and it had adequate room to comfortably move around. Testing for contextual conditioning: Twenty-four hours after training, each rat was placed in the same conditioning chamber as used for training. Freezing behavior in both novel and conditioning chambers was recorded. No sound or foot-shock was applied on test day. Freezing was measured using the inbuilt automated software (FreezeFrame 3.0, Coulbourne Instruments, Holliston, MA).

Experimental design

Rats were divided into four experimental groups (n = 7-12 rats in each group): group i. saline-treated non-stressed rats (sal no stress), group ii. alcohol-treated non-stressed rats (alc no stress), group iii. saline-treated rats subjected to stress (sal+RUPS), group iv. alcohol-treated rats subjected to stress (alc+RUPS). Rats were treated daily with alcohol or vehicle 30 min prior to the first stress exposure of the day. On day1 of stress (no morning stress), rats were treated at the same time as on the other days. No alcohol or vehicle was administered on the behavioral testing day.

Data Analysis:

Two-way or one-way ANOVA followed by post hoc tests was used to analyze the data (Prism5, GraphPad, La Jolla, CA). The level of significance was set at p<0.05.

Results

Adolescent rats tested for freezing in modified chamber:

Groups of rats were exposed daily to RUPS and treated with vehicle or alcohol for four days. Controls included non-stressed saline-treated and alcohol-treated rats. Rats were trained in the fear conditioning paradigm, and 24 h later placed in a modified chamber, and percent freezing was recorded (Fig. 1). A two-way ANOVA did not reveal any stress-by-drug interaction (F[1,29] = 0.03, p = 0.85), and there was no effect of stress (F[1,29] = 0.024, p = 0.878). There was significant treatment effect (F[1,29] = 13.12, p = 0.0011), but posthoc comparisons (Bonferroni test) did not show any difference between groups.

Figure 1:

Percent freezing in adolescent rats exposed to 4 days of unpredictable stress (RUPS) and daily treatment of saline or alcohol (for details see Materials and Methods). Controls included non-stressed rats treated with vehicle or alcohol. Rats were trained in the fear conditioning paradigm consisting of two cycles of US-CS pairings with an inter-trial interval of 90 sec. Twenty-four hours after training, rats were placed in a modified chamber; no foot-shock or sound stimulus was given on the test day. Percent freezing was recorded. A two-way ANOVA revealed no stress-by-drug interaction and no stress effect. There was significant treatment effect, but posthoc comparisons (Bonferroni test) did not show any significant difference between groups.

Contextual fear conditioning in adolescent rats subjected to repeated stress and alcohol treatment:

Stressed and non stressed rats treated with alcohol or vehicle were trained in the conditioning chamber, and 24 h later tested for contextual freezing (Fig. 2). A two-way ANOVA showed significant stress and drug interaction (F[1,36] = 6.19, p = 0.0176). There were significant effects of stress (F[1,36] = 6.889, p = 0.0126), and treatment (F[1,36] = 113.6, p < 0.0001). There was no difference in percent freezing between saline-treated non-stressed rats and stressed rats (78.33±4.63% and 79.05±5.76%, respectively). Compared to saline-treated non-stressed rats (78.33%±4.63%), alcohol-treated non-stressed rats (9.08±3.00%) showed significantly less freezing (p<0.001). Alcohol-treated stressed rats froze (36.01% ± 6.69%) significantly more (p<0.01) than alcohol-treated non-stressed rats (9.08±3.00%).

Figure 2:

Contextual freezing in adolescent rats exposed to 4 days of RUPS. Groups of stressed and non-stressed rats were treated daily with alcohol or saline. Rats were trained in the fear conditioning paradigm, and 24 h later tested for contextual freezing. Vehicle-treated stressed rats did not differ from vehicle-treated non-stressed controls. Alcohol significantly reduced freezing in non-stressed rats. Stressed rats treated with alcohol showed significantly less freezing than vehicle-treated stressed rats but froze significantly more than alcohol-treated non-stressed rats. ***p<0.001 compared to their respective vehicle-treated controls, ^$$p<0.01 compared to alcohol-treated non-stressed rats

Discussion

The present study was undertaken to investigate the effects of repeated unpredictable stress (RUPS) on memory formation in alcohol-treated female adolescent rats, with the premise that compared to RUPS only or alcohol only rats, alcohol-treated rats exposed to unpredictable stress would show greater memory impairment. In non-stressed adolescent rats, alcohol alone caused significant memory impairments. This finding is in line with previous studies from our laboratory [7,8,11]. There were two new interesting findings. The first one was that vehicle-treated RUPS adolescent rats did not differ in contextual freezing from vehicle-treated non-stressed rats. The second major finding was that alcohol-induced memory impairment was attenuated in stress exposed rats, suggesting partial reversal of alcohol-induced memory impairment by stress exposure.

Stress and freezing behavior in a novel environment:

When adolescent RUPS rats were trained in the fear conditioning paradigm and tested 24 h later in a modified chamber that was different from the conditioning chamber, without any sound stimulus or foot-shock, they froze less than when placed in the conditioning chamber (46.54±3.36% vs 78.33±4.64%, respectively). Therefore, when tested in a non-aversive environment (modified chamber), RUPS exposure had minimal effects on freezing behavior in both vehicle-treated and alcohol-treated rats. This suggests that exposure to stress with or without alcohol treatment, did not affect nonspecific fear.

Stress and contextual memory formation:

Contextual freezing in vehicle-treated RUPS rats did not differ from vehicle-treated non-stressed rats. There could be several reasons why contextual freezing in vehicle-treated RUPS rats froze as much as non-stressed vehicle-treated rats. One reason could be that the contextual fear conditioning paradigm may not be a sensitive behavioral readout to measure stress manipulation on learning and memory. This does not seem to be a valid argument since contextual freezing was impaired in alcohol-treated stressed rats. Another reason may be that four days of unpredictable stress was insufficient to alter contextual memory. That too appears to be unlikely since alcohol-treated stressed rats differed significantly from alcohol-treated non-stressed rats. A more plausible explanation is that adolescent rats were resistant to any stress-induced memory impairment.

Adolescent rats were subjected to a modified RUPS paradigm as previously described for adult animals [12]. Adult animals subjected to chronic variable stress exhibit alterations in memory [12,13,14]. Unlike adult rats, adolescent rats subjected to stress in the present study did not show any impairment in hippocampal memory. This finding is in agreement with other studies [15,16]. Adolescent animals exposed to unpredictable stress for eight days did not show any alteration in the performance of memory tasks [16]. Subjecting adolescent rats to even longer periods of variable stress, i.e. four weeks, did not impair spatial memory [15]. Thus, hippocampal memory formation in adolescent rodents appears to be resistant to stress. This suggests that adolescent rats may be resilient to stress-induced cognitive deficit. RUPS exposure in adolescent rats may be important in buffering, mitigating or moderating the effects of stress, and help in the development of coping mechanisms. Effects of the four-day RUPS protocol used in the present study has not been studied on memory acquisition in adult animals. Future studies will determine whether this stress paradigm alters contextual freezing in adult rats. In humans, several studies have reported that adverse stressful events or environments during early life promote resiliency to stress [17,18].

While a large literature exists on the mechanisms underlying the negative effects of stress on an individual’s physiology and behavior, the biological basis of stress resiliency remains relatively unknown. Biological substrates, including structural architecture, gene pathways, epigenetic factors, that determine stress resiliency in adolescent animals needs to be investigated.

Combined stress exposure and alcohol treatment on contextual memory.

Clinical research has shown that stress during adolescence increases risk for drinking [19]. In adolescent rodents also repeated stress has been reported to increase voluntary alcohol intake [20], although the rewarding effects of alcohol appears to be reduced [21]. Adolescent mice exposed to daily foot-shock stress for several days exhibited attenuation of alcohol-induced conditioned place preference (CPP), indicating reduced sensitivity to alcohol’s pleasure effect [21]. Whether stress-induced increased drinking affects hippocampal learning and memory needs to be examined.

The present study has shown for the first time that stress may be able to buffer alcohol’s negative consequences on cognitive functioning. These results raise several interesting issues such as sex-differences in the blunting of alcohol’s biological effects by stress, permanency of stress-induced reduction in alcohol-induced memory impairment, whether effects of stress exposure on hippocampal memory function are unique to adolescence or do similar changes occur in older animals, and if stress-induced buffering of alcohol’s cognitive damages are specific to hippocampus-related memory functions or are memory functions associated with other brain regions that undergo maturation during adolescence such as the prefrontal cortex (working memory) or amygdala (cued fear) also affected. Some of these possibilities are currently under investigation.

Mechanisms underlying the reversal of adolescent alcohol-induced contextual freezing following a relatively short period of repeated variable stress exposure, remain to be examined. Several systemic and neural modulators are known to contribute to alterations in stress-induced hippocampal behavior such as insulin-like growth factor-1 (IGF-1), cathepsin B, osteocalcin, brain-derived neurotrophic factor (BDNF), fibroblast growth factor2 (FGF2) and endocannabinoids [see review 22]. Stress has been shown to be associated with increased levels of IGF-1 [23], and lower levels of BDNF [24] and FGF2 [25]. Whether IGF-1, BDNF, FGF2 are important mediator(s) in adolescent stress-induced cognitive insensitivity to alcohol will be examined in the future.

Conclusion:

To summarize, exposure to repeated unpredictable stress did not alter contextual fear conditioning in adolescent rats. Alcohol significantly reduced contextual fear memory in non-stressed rats. Alcohol-treated RUPS rats showed significantly better memory than alcohol-treated non-stressed rats. Together, these data suggest (i) resiliency to stress-induced memory impairment in adolescent rats, and (ii) RUPS exposure causes blunting of alcohol’s negative effects on contextual fear conditioning.