The impact of low to moderate chronic intermittent ethanol exposure on behavioral endpoints in aged, adult, and adolescent rats

Accepted Manuscript The impact of low to moderate chronic intermittent ethanol exposure on behavioral endpoints in aged, adult and adolescent rats Douglas B. Matthews, Meredith R. Watson, Kimberly James, Abigail Kastner, Amelia Schneider, Guy Mittleman PII:

S0741-8329(18)30217-9

DOI:

https://doi.org/10.1016/j.alcohol.2018.11.005

Reference:

ALC 6877

To appear in:

Alcohol

Received Date: 25 July 2018 Revised Date:

13 November 2018

Accepted Date: 16 November 2018

Please cite this article as: Matthews D.B, Watson M.R, James K., Kastner A., Schneider A. & Mittleman G., The impact of low to moderate chronic intermittent ethanol exposure on behavioral endpoints in aged, adult and adolescent rats, Alcohol (2018), doi: https://doi.org/10.1016/j.alcohol.2018.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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THE IMPACT OF LOW TO MODERATE CHRONIC INTERMITTENT ETHANOL EXPOSURE ON BEHAVIORAL ENDPOINTS IN AGED, ADULT AND ADOLESCENT RATS

Schneider1 and Guy Mittleman2 1

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Douglas B Matthews1, Meredith R Watson1, Kimberly James1, Abigail Kastner1, Amelia

Department of Psychology, University of Wisconsin – Eau Claire and 2Department of

Address all correspondence to:

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Douglas B Matthews, PhD Department of Psychology University of Wisconsin – Eau Claire Eau Claire, WI 54701 [email protected] 715.836.2119

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Psychological Science, Ball State University

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Abstract The average age of the population in the United States and other countries is increasing. Understanding the health consequences in the aged population is critical. Elderly individuals

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consume ethanol, often at elevated rates, and in some cases in a binge episode. The present study sought to investigate if binge like ethanol exposure in aged male rats produced differential health and behavioral effects compared to adult male and adolescent male rats. Subjects were

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exposed to either 1.0 g/kg or 2.0 g/kg ethanol every other day via intraperitoneal injection for 20 days and tested on a variety of behavioral measures and body weight. Binge like ethanol

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exposure produced differential effects on body weight between aged and adolescent and adult rats. In addition, aged rats had significantly longer loss of righting reflex and demonstrated a trend toward tolerance following the 2.0 g/kg exposure. No significant effects on anxiety-like behavior as measured by open arm entries, depressive-like symptoms as measured by immobility

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in the forced swim test or cognitive performance as measured by latency and pathlength in the Morris water maze were found. These results demonstrate that aged animals are differentially sensitive to the impact of chronic intermittent ethanol exposure in some, but not all behaviors.

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Future research is needed to understand the mechanisms of these differential effects.

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Keywords: Aged, Ethanol, Tolerance, Loss of Right Reflex, Body Weight

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Recent population predictions indicate that the number of Americans aged 65 and older will double from 46 million to 98 million people by 2060. In addition, it is predicted that by

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2060 this age demographic will account for up to 24% of the population in the United States (Ortman, Velkoff, & Hogan, 2014). Aging of the general population is not restricted to the

United States but is also occurring in many developed and developing countries (Ortman et al.,

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2014). This rapid increase in the aging population will place a strain on both the health care and social care in the United States (Fact sheet, Population Reference Bureau, 2016) potentially

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producing what has been termed a “silver tsunami” (Tampi, Tampi, & Durning, 2015). Consequently, it is important to investigate and understand the impact of risk factors that influence the state of health of elderly individuals.

One risk factor negatively impacting the health of elderly individuals is high levels of

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ethanol consumption. Recent demographic data reveal that over 33% of people aged 65 years or older engage in ethanol consumption, with 10% of men engaging in heavy ethanol drinking (Breslow, Faden, & Smothers, 2003). The increase in heavy ethanol use by people older than 65

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years of age is highlighted by data demonstrating that more than 14% of men and almost 5% of women engaged in binge ethanol drinking in the last year (Blazer & Wu, 2009). In fact, recent

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work has suggested the number of elderly engaging in ethanol use is increasing (Breslow, Castle, Chen, & Graubard, 2017). Furthermore, 1-3% of elderly suffer from a diagnosed alcohol use disorder (AUD) (Blazer & Wu, 2009), although it is suspected that the diagnosis of an AUD in the elderly population is underreported (Caputo et al., 2012). Considering that heavy ethanol use can increase the risk of several diseases including diabetes, high blood pressure, congestive heart failure and memory problems (Stevenson, 2005; Thomas and Rockwood, 2001; see Rehm et al.,

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2009 for review), coupled with the fact that ethanol is often reported to produce larger effects in elderly adults compared to younger adults (Brower, Mudd, Blow, Young, & Hill, 1994; Kraemer, Mayo-Smith, & Calkins, 1997; Campbell, Gabrielli, Laster, & Liskow, 1997;

ethanol consumption in the elderly population.

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Menninger, 2002), it is crucial to understand the health consequences of binge or excessive

Investigating potential negative impacts of ethanol use in the elderly has largely been

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hampered by the lack of suitable animal models. Previous work with younger animals has

shown that chronic ethanol exposure results in a variety of alterations in behavior. For example,

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chronic ethanol exposure can increase anxiety-like behaviors (for example see Novier et al., 2015), increase depressive-like behavior (Walker et al., 2010) and impair cognitive performance on some tasks (Obernier et al., 2004). A small number of studies has used aged rats as subjects, revealing differences in the response to an ethanol challenge in aged rats compared to either

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adult or adolescent rats. In comparison to adult animals, aged rats appear more intoxicated and have greater withdrawal scores following 14 days of liquid ethanol diet (Wood, Armbrecht, & Wise, 1982). In addition, aged Sprague-Dawley rats given an acute ethanol challenge are

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significantly more sensitive to the ataxic effects of ethanol compared to either adult or adolescents rats (Van Skike et al., 2010; Novier, Van Skike, Diaz-Granados, Mittleman, &

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Matthews, 2013; Ornelas, Novier, Van Skike, Diaz-Granados, & Matthews, 2015), and are significantly more impaired on spatial memory tasks compared to adult rats (Novier et al., 2013). If animals are exposed to ethanol in a chronic fashion, aged Sprague-Dawley rats show more general anxiety-like behaviors on the elevated plus maze compared to adult rats (Novier, Ornelas, Diaz-Granados, & Matthews, 2016). Furthermore, chronic ethanol exposure in aged rats does not produce tolerance to a moderate ethanol dose challenge (2.0 g/kg ethanol, i.p.) as

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assessed via the aerial righting reflex (Matthews & Mittleman, 2017). If body weight is used as a proxy for general health, chronic ethanol exposure results in a significant reduction in body weight in aged rats compared to adolescent and adult rats, suggesting that chronic ethanol in

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aged populations can lead to potential health issues (Matthews & Mittleman, 2017).

While research suggests that ethanol exposure in aged animals results in different

behavioral responses compared to either adolescent or adult animals, little is known about

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mechanisms that underlie these differences. Previous work has demonstrated that ethanol levels obtained from tail-blood 30 to 60 minutes following intraperitoneal administration are similar

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between adolescent, adult and aged rats suggesting that a simple explanation such as blood ethanol levels at the time of testing cannot account for the differential behavioral results (Novier et al., 2013; Matthews & Mittleman, 2017). One potential mechanism underlying the differential acute effects of ethanol in aged rats is altered neuroimmune function and cytokine expression in

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the hippocampus (Gano, Doremus-Fitzwater, & Deak, 2017), where such an age specific effect may underlie the exacerbated impaired spatial memory deficits found in aged animals following an ethanol challenge (Novier et al., 2013).

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Many of the people who suffer from an AUD are diagnosed in adulthood, however, a growing proportion of people are being diagnosed with an AUD later in life. Approximately

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33% of those suffering from an AUD are classified as late onset alcoholics (see Novier, DiazGranados, & Matthews, 2015 for review). In addition to knowing little about the negative impact of ethanol in elderly populations in general, even less is known of the negative manifestations of late onset alcoholism. Initial studies designed to investigate chronic ethanol use in aged animals compared to adult animals used ethanol liquid diet as the exposure paradigm. However, adult animals consumed significantly more ethanol and had higher blood ethanol

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levels (BEC) than aged rats, thereby hampering direct comparisons between the ages (Novier et al., 2016). In the current project, we sought to capitalize on our recent method (Matthews & Mittleman, 2017) where aged, adult and adolescent rats were injected intraperitoneally with

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ethanol in a chronic, binge-like fashion, a procedure that produces similar initial blood ethanol levels (Matthews & Mittleman, 2017). To this end, we investigated if low or moderate chronic intermittent ethanol exposure (CIE) in aged rats differentially impacted a variety of behavioral

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endpoints compared to CIE in adult and adolescent animals. Based on previous research

following long-term, high dose ethanol exposure we predicted that aged animals treated with

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ethanol chronically would show increases in anxiety-like behaviors and depressive-like behaviors and altered learning and memory. However, considering the current studies use low dose ethanol exposure, it is possible that not all behaviors will be significantly affected. Materials and methods

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Subjects: Eighty-six male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) were used to investigate the effect of chronic intermittent ethanol exposure on body weight, anxiety measures (elevated plus maze), depressive-like symptoms (forced swim task), loss of

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righting reflex (LORR) and non-spatial and spatial memory in the Morris water maze. Subjects were classified into one of three different age cohorts, including early-adolescent (Shipped at PD

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28, n = 30, average body weight 107 grams at the start of the experiment), young adult (Shipped at PD 70, n = 26, average body weight 317 grams at the start of the experiment) and aged (shipped at PD ~ 18 months, retired breeders, n = 30, average body weight 578 grams at start of the experiment). Animal care procedures followed the guidelines of the University of Wisconsin - Eau Claire IACUC. Food and water were provided ad libitum except for the saline control group that had food access yoked to the high dose ethanol group in order to maintain similar

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body weights (see below). Animals were housed two animals per cage in an Ecoflo system (Allentown Caging, Allentown, New Jersey) on a 12:12 light:dark cycle (lights on at 6:00 am; lights off at 6:00 pm). All animal procedures occurred between 9:00 am and noon on test days.

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Chronic Intermittent Ethanol exposure (CIE): Early-adolescent, adult and aged

animals were randomly divided into one of two ethanol groups or one control group and received either ethanol (1.0 g/kg [early-adolescent n = 10 , adult n = 8 , aged n = 10] or 2.0 g/kg [early-

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adolescent n = 10 , adult n = 10 , aged n = 10 ]) or saline (saline injection amount was matched in volume to the 2.0 g/kg ethanol injection amount [early-adolescent n = 10 , adult n = 8 , aged n

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= 10]). Ethanol, 20% v/v, was administered via intraperitoneal injection every 48 hours for 20 days, for a total of 10 intoxications and withdrawals as previously described (Matthews, Tinsley, Diaz-Granados, Tokunaga, & Silvers, 2008). During treatment, all animals in the CIE exposure groups had unrestricted access to food and water; all animals that received saline were weight

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yoked to the average 2.0 g/kg CIE-treated rats’ weight of the same age to control for ethanolinduced weight suppression. Injections begin on PD 30, PD 72 or ~PD 18 months, respectively, for each condition, commencing two days following arrival in the laboratory. Animals were

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weighed daily as an indirect index of general health while CIE was ongoing. Blood ethanol concentration: Following the first CIE treatment day, animals had their

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tail nicked and approximately a 5 ul blood sample was collected 30 minutes following injection, spun down to plasma and blood ethanol concentrations determined using an AM-1 Analox machine (North Yorkshire, United Kingdom). Elevated Plus Maze: Twenty-four hours after the final ethanol-exposure, anxiety-like

behaviors was measured on an elevated plus maze (Any-maze, Stoelting Co., Wood Dale IL 60191) located in a behavioral room isolated from animal caging and housing. The apparatus

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was elevated 50 cm from the ground and consisted of four arms 50 cm in length and 10 cm wide. The walls of the closed arms were 40 cm in height and located at opposing sides of the maze. Light levels were approximately 12 LUX in the closed arms and 74 LUX in the open arms.

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Animals were moved to the testing room 10-20 minutes prior to experimentation for acclimation. Each animal was placed in the central location facing an open arm and allowed 5 minutes to explore the maze. Each animal was videotaped and data was analyzed later by two research

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teams of two experimenters blind to the CIE condition. The apparatus was wiped clean with a dilute ethanol solution between trials. The data was averaged across scores of the two teams for

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open arm entries, closed arm entries, total movement (open arm entries + closed arm entries + middle area entries), percent open arm entries (open arm entries divided by open arm entries + closed arm entries*100) and percent open arm time (open arm time [in seconds] divided by 300*100). In order to be defined as an entry all four paws had to be within a maze compartment.

movement.

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Separate ANOVAs were conducted on open arm entries, percent open arm entries and total

Forced Swim Test: Eight days after the last CIE treatment day, depressive-like behavior

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was assessed via a modified version of the forced swim test (Slattery & Cryan, 2012; (Yankelevitch-Yahav, Franko, Huly, & Doron, 2015). Each subject first received a 10-min

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pretest followed 24 hours later by a 5 minute test session. The forced swim was conducted in an apparatus that was 20 cm in diameter and 45 cm high (Any-maze, Stoelting Co., Wood Dale IL 60191). The apparatus was filled with water to a height of 35 cm at 23 degree C and refilled between subjects. Each test session was videotaped and the subject was scored as swimming or immobile every 5 seconds by two teams of two researchers that were blind to the CIE condition

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of the subject. The number of immobile time bins was determined by the average mobile bin score of the two teams.

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Loss of Righting Reflex (LORR): Twenty-one days following completion of CIE treatment, the effect of a moderately high dose of ethanol on sleep-time was determined by monitoring the loss of righting reflex of each subject. Animals were tested in the morning

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(approximately the same time of injection of the previous CIE treatment) and were weighed and injected, i.p., with 3.0 g/kg ethanol. Animals were carefully monitored for loss of righting reflex

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and the time that the righting reflex was regained. Regaining righting reflex was defined as the animal successfully righting itself three times within 60 seconds. If an animal did not lose its reflex, its LORR was scored as zero.

Non-spatial, cue based, learning: Six weeks following completion of CIE treatment,

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non-spatial, cue-based learning was assessed using the visible platform version of the Morris Water maze (Morris, Garrud, Rawlins, & Okeefe, 1982). The tank used was six feet in diameter and the water level was approximately 24 inches deep. The tank was filled with water that was

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made slightly opaque with the addition of white tempera nontoxic watercolor paint. Animals were trained to swim from the four compass locations to a platform that protruded above the

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water surface by approximately 1.0 cm and was covered in black electrical tape. Animals received four trials per day for three days. The order of the start locations was constant while the location of the platform was changed every day to ensure animals could not use spatial cues to guide performance. The latency to the platform and the path-length was determined for each animal using Any-maze digital tracking software (Any-maze, Stoelting Co., Wood Dale IL 60191).

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Spatial, location based, learning: Between seven and eight weeks following completion of CIE treatment, spatial learning was assessed using the hidden platform version of

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the Morris Water maze (Morris et al., 1982). The apparatus used was the same as that used in the non-spatial, cue based learning task. However, in this task the escape platform was 1 cm below the water surface and the identifying black electrical tape was removed. In addition, the

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location of the escape platform was constant during training while the order of the start locations was randomly varied. Animals received four trails per day for three days. The latency to the

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platform and the path-length was determined for each animal via Any-maze digital tracking software (Any-maze, Stoelting Co., Wood Dale IL 60191). Figure 1 provides an overview of the experimental timeline.

RESULTS

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Effect of CIE on Body Weight at Different Ages

To investigate the effect of CIE at different ages on body weight we conducted several different analysis. First we investigated the effect of ethanol exposure during the CIE exposure

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period. In agreement with previous work (Matthews & Mittleman, 2017) we found that age and the CIE dose administered significantly interacted over the exposure period (Three way ANOVA

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with repeated measures (age of subject by CIE dose by CIE treatment day, F = 3.558, df (72,1314), p < 0.001), see Figure 2. To better understand the impact of CIE exposure and age on bodyweight we collapsed the data across the exposure period and analyzed the dose of ethanol given as a function of the age of the animals. Once again, we found that ethanol dose and age significantly interacted to impact bodyweight (Two Way ANOVA (age of subject by CIE dose), F = 22.59, df 4,73), p < 0.001). As expected, adolescent rats administered saline gained

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significantly more body weight than either adults or aged rats (Scheffe post hoc test, p < 0.05). Importantly, following either 1.0 g/kg or 2.0 g/kg ethanol exposure, the aged animals’ body weight was significantly different from both adolescent and adult animals (Scheffe post hoc text,

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p < 0.05). See Figure 3.

We also investigated the impact of CIE within each of the three age groups. To

accomplish this we conducted a series of Two-way ANOVAs using body weights from the first

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and the last days of treatment at each dose of ethanol (dose of ethanol by treatment day) for each age (adolescent, adult and aged rats). Table1 shows the results of these comparisons. In aged

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animals, the dose of ethanol administered interacted with the treatment day to affect body weights (Two Way repeated measures ANOVA, dose of ethanol by treatment day, F = 6.16, df(2,27) p < 0.01). Post hoc analysis revealed that no differences in body weight existed between doses on the first day of treatment for aged rats, but on the last day of treatment, animals

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administered 1.0 g/kg ethanol weighed significantly more than animals administered 2.0 g/kg (Sidak’ multiple comparisons, p < 0.05). The similarity in bodyweights on the last CIE test day indicates that the yoking procedure was successful in this age group.

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In adult animals, the dose of ethanol administered interacted with the treatment day to affect body weight (Two Way ANOVA with repeated measures, dose of ethanol by treatment

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day, F = 8.35, df(2,20), p <0.01). As expected, no difference in body weight was found on treatment day 1. On the last day of treatment saline animals weighed significantly less than either the animals administered 1.0 g/kg or 2.0 g/kg ethanol (Sidak’s multible comparisons, p < 0.05). The difference in bodyweights on the last day of CIE indicate yoking procedure was less successful in adult animals.

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Finally, in adolescent animals, the dose of ethanol administered interacted with the treatment day to impact body weight (Two Way ANOVA with repeated measures, dose of ethanol by treatment day, F = 138.4, df(2,27), p <0.001). As expected, no difference in body

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weight was found on treatment day 1. On the last day of treatment saline treated animals

weighed significantly less than animals administered 1.0 g/kg or 2.0 g/kg ethanol (Sidak’s

multiple comparisons, p < 0.01). The difference in bodyweights on the last day of CIE indicate

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yoking procedure was less successful in adolescent animals.

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Elevated Plus Maze:

Behavior on the elevated plus maze was hand scored from videotape by two teams of undergraduate researchers who were blind to CIE condition (age of animals was not possible to blind due to size of animals; inter-rater reliability for closed arm entries was r2 = 0.74 while open

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arm entries was r2 = 0.84). Consistent with previous research, animals that fell off the elevated maze during the five-minute test session were placed back on the maze to equate session time and experience, but their data were not included in the analysis (Walf & Frye, 2007). As a

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measure of anxiety, we analyzed the number of open arm entries, percent of open arm entries and percent open arm time as well as two measures of total movement. The CIE treatment did not

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produce an anxiogenic response in the ethanol treated animals, likely due to a low number of open arm entries in control animals. This was evidenced by non-significant effects on open arm entries (Two Way ANOVA, age of subject by CIE treatment, no main effect of CIE dose, age of subject or interaction, all p’s > 0.10), percent open arm entries (Two Way ANOVA, age of subject by CIE treatment, no main effect of CIE dose, age of subject or interaction, all p’s >

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0.10) and percent open arm time (Two Way ANOVA, age of subject by CIE treatment, no main effect of CIE dose, age of subject or interaction, all p’s > 0.10). There was a significant interaction of CIE and age in terms of total movement (Two-Way

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ANOVA, age of subject by CIE treatment, F = 3.203, df (4,61), p < 0.05 and significant main effect of age, F = 3.575, df (2,61), p < 0.05). Adolescent animals treated with saline had

significantly more closed arm entries compared to adult and aged animals treated with saline

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(Tukey post hoc test, p < 0.05 for both groups) while no other comparisons (age or CIE ethanol condition) were significant. The effect of CIE on movement was consistent when total

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movement (open arm + closed arm + center) was analyzed (Two-Way ANOVA, age of subject by CIE treatment, F = 2.71, df (4,61), p < 0.05).

Once again, adolescent animals treated with

saline had significantly greater total movement compared to both adult and aged animals treated with saline (Tukey post hoc test, p < 0.001 for both groups) while no other comparisons (age or

Forced Swim Test

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CIE ethanol condition) were significant. See Table 1.

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To investigate if CIE treatment at different ages altered depressive-like symptoms in animals we investigated the number of 5-sec bins where the animal was scored as being

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immobile. Scoring was accomplished by having two teams of two researchers blind to the CIE condition score the videotaped sessions, and then the average of their scores were used for analysis (inter-rater reliability was r2 = 0.26 for a measure that yields poor inter-rater reliability [Slattery & Cryan, 2012]). Age of the animal significantly impacted depressive-like symptoms. Regardless of treatment (ethanol or saline) older rats had significantly more 5-sec time bins scored as immobile compared to adolescent animals (Two Way ANOVA, age of subject by CIE

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dose, main effect of age, F = 3.88 df(2,66), p < 0.05; post hoc Tukey comparison, p < 0.05). See Table 1.

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Loss of Righting Reflex To investigate the impact of CIE treatment on the initial sensitivity of animals to a

moderately high dose of ethanol, we investigated the duration of the loss of righting reflex

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following a 3.0 g/kg ethanol injection. Aged animals were significantly more sensitive to the effects of ethanol while CIE dose was marginally significant at modifying this effect (Two Way

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ANOVA, age of subject by CIE dose, F = 2.51, df(4,62), p < 0.051; main effect of age, F = 12.18, df (2,62), p < 0.001). See Figure 4.

Non-spatial learning.

To investigate general learning ability in CIE treated animals, we first investigated non-spatial

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learning in the Morris water maze. The chronic intermittent ethanol dose did not interact with age of the animal or the day of training (Three way ANOVA, age of subject by CIE dose by day

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of training, F=1.08(8,118), p > 0.05). However, age of the subject significantly interacted with training day to impact latency to find the platform (Two way ANOVA, age of subject by day of

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training, F = 3.82, df (4,130); p < 0.01). Specifically on day one of training, adolescent animals performed better than both the adult and aged rats when performance was measured as latency (Tukey post hoc test, p < 0.01) while on training days 2 and 3, aged rats performed significantly worse than both adolescent and adult rats when latency was analyzed (Tukey post hoc test, p < 0.001). These age effects were mirrored by the analysis of swim pathlength (Two Way ANOVA, age of subject by day of training, F = 3.162, df (4,130); p < 0.05). On training day 1 adolescents swam a significantly shorter distance to the platform than adult or aged rats (Tukey

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post hoc test, p < 0.01) while aged rats swam significantly longer distances than adolescents or adults on training day 2 and day 3 (Tukey post hoc test, p < 0.001). This latter finding showed that the impairment in non-spatial learning in aged rats was not simply due to poor swimming

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ability. See Table 2. Spatial Learning

To investigate the impact of CIE at various ages on hippocampal dependent, spatial

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learning, we analyzed animals’ spatial learning over a short 3-day training period in the spatial version of the Morris water maze. Similar to results from the non-spatial learning task, CIE

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administration did not interact with either age of the animal or training days to impair learning in the spatial version of the Morris water maze as measured by latency to the platform (Three Way ANOVA, age of subject by CIE dose by day of training, p > 0.05). However, animals performed differently over the training days (Two way ANOVA, age of subject by day of

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training, F = 2.88, df(4,118), p < 0.05). Specifically, adolescent rats performed significantly better than adult or aged rats on training day 3 (Tukey’s post hoc test, p < 0.05). In addition, swim pathlength demonstrated a similar age effect over the training days (Two Way ANOVA,

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age of subject day of training, , F = 6.85, df(4,106), p < 0.01). Once again, adolescent rats had shorter pathlengths compared to adult and aged on training day 3 (Tukey’s post hoc test, p <

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0.05). See Table 2.

Blood Ethanol Levels

Blood ethanol concentrations were investigated from plasma samples drawn 30 minutes

following the first ethanol exposure period. At this time point there was not a clear age dependent affect, in that adult rats had higher ethanol levels than adolescent rats but not aged

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rats. Blood ethanol levels in adolescent and aged rats did not differ. As expected, blood ethanol levels differed depending upon dose of ethanol administered (Two way ANOVA, age of subject

df(1,50), p < 0.0001). See Figure 5. DISCUSSION

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by CIE dose, main effect of age, F = 3.6, df(2,50), p < 0.05; main effect of dose, F = 61.19,

As the average age of the population increases, understanding risk factors that can

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compromise health is critical. One risk factor that exists in the aged demographic is increasing ethanol intake and often binge-like consumption that has resulted in a segment of the elderly

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population being diagnosed with an AUD. Little is currently known about the interaction of age and binge like ethanol exposure in the elderly population due to a lack of established animal models including baseline behavioral data comparing ethanol exposure in elderly animals to other, better studied populations such as adult or adolescent animals. The current work extends

behavioral measures.

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the understanding of the interaction of aging with binge like ethanol exposure on a variety of

The impact of chronic binge-like ethanol exposure in aged rats compared to adult or

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adolescent rats produced differential changes that were dependent on the behavioral measure investigated. One of the most striking differential impacts of CIE in aged rats was the change in

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body weight. Aged rats general body weight was significantly reduced following CIE while the body weight of adolescent animals continued to increase under the CIE protocol. Such a reduction in body weight in aged rats is problematic considering that reductions in body weight can be a significant heath factor in elderly individuals (Miller & Wolfe, 2008). The differential impact of CIE on body weight has been previously shown in our laboratory (Matthews & Mittleman, 2017). However, the current work used lower ethanol doses than our previous study

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and still found a significant reduction in body weight in the aged rats. The current study used a low dose of ethanol, 1.0 g/kg, for the CIE dose, a dose that produced an average BEC of 113.9 mg/dl 30-minutes following injection. While this level of ethanol is above the legal limit of

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intoxication in the United States, it is not a significantly elevated level and one that would be easily obtainable in elderly people. Given that AUDs are often under-reported in the elderly population, this health concern may be missed by health care providers. As such, additional

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training and care by the health system on elderly drinking is needed.

A variety of age-related factors may play a role in the reduced body weight of aged

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animals produced by CIE. Adolescent rats have less hangover-like systems compared to adult rats (Brasser & Spear, 2002; Varlinskaya & Spear, 2004) and adolescent and adult rats have less ethanol-induced motor ataxia compared to aged rats (Novier et al., 2013; Ornelas et al., 2015; Van Skike et al., 2010; Novier et al., 2016). Consequently, adolescent and perhaps adult rats

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may be able to engage in feeding behavior faster following ethanol exposure. However, to the best of our knowledge, it is currently unknown if aged rats have greater hangover-like symptoms compared to adolescent or adult rats, so this possibility deserves attention. Furthermore,

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conditioned taste aversion, a unique form of long lasting learning that can impact drugs of abuse (Verendeev & Riley, 2012), develops less in adolescent animals following ethanol exposure than

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adult animals (Holstein, Spanos, & Hodge, 2011). This lack of taste aversion may also lead to more food intake in adolescent animals following ethanol exposure. Finally, aged rats exposed to CIE may absorb nutrients differently from the gut compared to younger animals following CIE.

The experimental design used a yoking strategy to ensure subsequent measures of anxiety-like behaviors, depressive-like or cognitive ability would not be impacted by CIE-

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induced reductions in body weight compared to saline-treated animals. Previous work has demonstrated that CIE exposure will reduce body weight (Silvers et. al., 2003; 2006) and this effect is greater in aged animals (Matthews & Mittleman, 2017).

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In the current research, the weight of the aged animals administered saline were

successfully yoked to the weight of the aged animals administered 2.0 g/kg ethanol. As a

consequence of this yoking procedure adult and adolescent saline treated animals weighed less

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than the adult and adolescent 1.0 g/kg ethanol and 2.0 g/kg ethanol treated animals. While not ideal, we are confident for the following reasons that any resultant effect of CIE is not due to

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reduced body weight in the saline animals. First, the average difference in body weight between saline treated adolescent animals and CIE treated adolescent animals or saline treated adult animals and CIE treated adult animals is small and likely does not negatively impact the behaviors measured in the current project. This conclusion is supported by similar behavioral

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outcomes on a variety of the experiments. Second, aged animals administered 2.0 g/kg ethanol were successfully body weight yoked to the corresponding aged animals treated with saline. Given the majority of the significant findings are in the aged animals, we have confidence these

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effects are not due to differential body weight. Third, when comparing across ages, the differential effect of CIE on bodyweight is driven primarily by differences in animals

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administered 2.0 g/kg; aged animals lost body weight while adolescent animals increased body weight. While these findings provide support for the data reported in the current manuscript, it is important to note the current work did not utilize an ab libitum free feeding saline group. Future research should include this type of condition to better understand the impact of CIE on bodyweight across the lifespan.

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Previous research has shown that aged rats are extremely sensitive to the ataxic effects of acute ethanol (Van Skike et al., 2010; Matthews & Mittleman, 2017). The loss of righting reflex has been used to demonstrate a very large response to ethanol in aged rats following a

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moderately high ethanol dose (3.0 g/kg). The current work sought to extend these findings by determining if low to moderate CIE would mitigate the increased effect in aged rats to a similar 3.0 g/kg ethanol injection. As expected, aged rats had significantly longer sleep times following

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the 3.0 g/kg ethanol injection in the LORR test compared to either adult or adolescent animals. In addition, it appears that moderate dose CIE may produce a tolerance-like effect on LORR in

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aged rats in that the interaction of CIE dose and LORR was marginally significant (p<0.051). In support of this, it should be noted that aged rats administered 2.0 g/kg CIE slept 79% less than aged rats administered saline during the CIE treatment period. The impact of these effects is at least three-fold. First, the repeatedly demonstrated effect that aged rats are significantly more

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impaired by ethanol compared to adult or adolescent rats suggests that primary health care providers should specifically warn older patients of the risk of ethanol use and ataxia. Secondly, 0.08 mg/dl is the legal limit of intoxication for operating an automobile. However, given the fact

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that aged animals (Wood & Armbrecht, 1982; Van Skike et al., 2010; Novier et al., 2013; Ornelas et al., 2015; Matthews & Mittleman, 2017) as well as aged humans (see Squeglia,

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Boissoneault, Van Skike, Nixon, & Matthews, 2014 for review) are significantly more impaired by ethanol it is likely that the level of intoxication should not be standard across all ages and should perhaps be lower in aged individuals. Finally, blood ethanol levels at the time of testing cannot be a general explanation for the disparate effect of ethanol found in the present study. Several studies including the present work have demonstrated that age related differences in BEC do not occur following an intraperitoneal injection when plasma is collected 30 to 45

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minutes following injection. In addition, Ornelas et al., (2015) showed that following 3.0 g/kg ethanol injection, i.p., BEC levels were similar at 60 and 120 minutes later (the critical time for the LORR studies reported here). As such it is reasonable to conclude that some factor(s) other

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than just blood ethanol levels impacts the LORR in aged rats. To more fully conclude that aged rats are indeed more sensitive to the sleep time effects of high dose ethanol, blood ethanol concentrations at waking can be assessed. A recent study supports the data in the current

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manuscript by demonstrating that high dose ethanol (3.0 g/kg and 3.5 g/kg ethanol) result in longer LORR sleep times and lower blood ethanol levels at waking (Perkins et al., in press).

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Future research should expand on these findings as it relates to ethanol tolerance in LORR. Chronic intermittent ethanol exposure did not result in an increase in anxiety-like behaviors, as measured in the plus-maze, when the anxiety state was determined approximately 24 hours following the last CIE treatment. Specifically, CIE animals had similar open arm

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entries, percent open arm time and percent open arm entries as control treated animals and this effect was not modified by the age of the animal. This was unexpected considering early studies have shown that CIE exposure (via liquid diet) increased anxiety-like behavior on the plus maze,

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and this increase was largest in aged animals (Maier & Pohorecky, 1989; Novier et al., 2016; for review see Kliethermes, 2005). However, when comparing between adolescent and adult rats,

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CIE treatment does not appear to produce consistent increases in anxiety-like behaviors (Doremus, Varlinskaya, & Spear, 2004) so overarching conclusions should be drawn with care. Several possible reasons exist why the CIE treatment did not cause anxiety-like behavior

in the current study. First, treatment and control animals, did not venture frequently onto the open arms of the maze. As such a floor effect likely exists making it difficult to demonstrate an anxiety-like state, even if one existed. This possibility is supported by previous work showing

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that higher numbers of open arm entries are associated with demonstration of anxiety-like behavior in the plus maze following CIE exposure (Van Skike, Diaz-Granados, & Matthews, 2015). Importantly, open arm entries in the control animals were significantly greater in the Van

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Skike study (~40%) compared to the present data (~20%). Secondly, the doses of ethanol used in the CIE treatment were lower (1.0 g/kg and 2.0 g/kg) and may not have been sufficient to produce an anxiety-like state. Third, the length of CIE exposure may not have been sufficient to

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produce anxiety. Previous work demonstrating CIE can produce anxiety-like behavior in aged rats used liquid diet for several weeks instead of the 10 acute exposures in the current study

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(Novier et al., 2016). Fourth, food restriction in the saline animals during the CIE exposure period could be stressful. Future studies should include a free-feeding saline control condition to monitor this potential confound. Fifth, there was low movement between arms on the plus maze during the test and low movement can confound interpretation of anxiety-like behavior in this

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test. Finally, the plus maze might not be the best procedure and apparatus to use when studying anxiety in aged rats. Following previous research norms, if an animal fell from the plus maze their data were not used; aged rats (4 rats) fell off the maze more often than adult (3 rats) or

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adolescent rats (0 rats).

Previous work suggests that total movement in the elevated plus maze is similar between

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male adolescent and male adult rats in terms of the percent of variance accounted for in a factor analysis procedure (Doremus et al., 2004; Doremus, Varlinskaya, & Spear, 2006). In the current study we found that adolescent animals have higher levels of movement as measured by either the number of closed arm entries or total movement than adult or aged animals on the elevated plus maze and this increased activity is significantly reduced following CIE exposure. This supports previous work in our laboratory suggesting that CIE exposure can reduce locomotor

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activity in the elevated plus maze (Van Skike et al., 2015). Previous work has demonstrated that acute ethanol exposure can increase locomotor activity in the elevated plus maze in adolescent rats (Acevedo, Pautassi, Spear, & Spear, 2013) suggesting this effect might show tolerance

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following CIE exposure.

The forced swim task was used to determine if CIE exposure produces age-dependent increases in immobility, a putative measure of depressive-like behavior. Surprisingly, low to

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moderate CIE exposure in the current study did not increase immobility behavior in any age group, although previous studies have shown that ethanol exposure can increase immobility in

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both adolescent (Ehlers, Criado, Wills, Liu, & Crews, 2011; Slawecki & Roth, 2004) and adult (Walker et al., 2010) rats. Several methodological factors may explain these disparate results including: significantly longer ethanol exposure periods (Ehlers et al., 2011); 14-hr long daily vapor exposures (Ehlers et al., 2011; Slawecki & Roth, 2004; Walker et al., 2010) as opposed to

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every other day i.p. injections; the rat strain used as subjects in the study (Wistar [(Ehlers et al., 2011; Slawecki & Roth, 2004; Walker et al., 2010] compared to Sprague Dawley [current study]). The current work did find a significant impact on immobility as a function of age, with

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older rats demonstrating significantly greater floating behavior compared to adolescent rats, a finding that is in agreement with previous research (Turner et al., 2012).

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Chronic intermittent ethanol exposure did not impair either non-spatial or spatial learning

in the Morris water maze, although performance on both tasks was impaired in aged animals compared to younger animals. It is not surprising that non-spatial learning was similar between both CIE treated and control treated rats considering several studies have documented ethanol’s lack of effect on this task (for review see Novier et al., 2015). However, it was somewhat surprising that CIE did not impair spatial learning.

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Spatial learning in the Morris water maze is a task that is hippocampal dependent (Morris et al., 1982) and several studies from a variety of laboratories have shown that acute and chronic ethanol exposure can impair spatial learning (Matthews, Simson, & Best, 1995; Matthews, Ilgen,

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White, & Best, 1999; Acheson, Ross, & Swartzwelder, 2001; Obernier, White, Swartzwelder, & Crews, 2002; Novier et al., 2016)) . Several factors might explain the lack of impairment in the current task. First, low to moderate levels of ethanol were used in the CIE treatment period and

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other chronic studies have used either higher ethanol doses or longer exposure periods.

Secondly, animals’ spatial learning was not tested until almost 9 weeks after the conclusion of

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the CIE exposure period. This long delay between CIE exposure and testing might have mitigated any impairment produced by ethanol.

Understanding the impact of chronic binge-like ethanol exposure in the elderly has been hampered due to few studies directly comparing the impact of ethanol exposure in this age to the

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impact of ethanol in other, younger, ages. The current work clearly demonstrates that chronic intermittent ethanol exposure differentially impacts aged animals compared to young adult or adolescent rats on some health factors and behavioral endpoints. However, this work also

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highlights several additional lines of research that need to be undertaken to better understand this important societal question.

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First, the doses of ethanol used in the chronic intermittent exposure paradigm are low to

moderate doses. While these doses do produce blood ethanol concentrations greater than the legal limit of intoxication, the resultant BEC levels are not excessively high. Therefore it is likely that increased impairments would be found if animals were treated with higher doses of ethanol during CIE. In addition, blood ethanol levels should be assessed at additional time points rather than simply on the first day of exposure in order to determine if differential

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metabolic tolerance occurs. Secondly, it is still unclear why aged rats consistently lose body weight during the CIE treatment at levels greater than adult animals or adolescent animals (which show increases in body weight). Previously we suggested several behavioral effects

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(differential hangover effects, taste aversion) that may impact bodyweight in aged rats. In

addition, it is possible that chronic intermittent ethanol in aged rats alters food absorption from the gut or increases nutrient elimination via urine and/or feces. This effect could represent a

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significant health issue in elderly people and needs additional research. Furthermore, the vast majority of the research on chronic ethanol and body weight in aged rats has only used male

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subjects. Given the fact that females live longer than males on average in the United States and other countries it is imperative the future studies be expanded to include female rats as subjects. Third, the lack of an anxiogenic effect is surprising and could be influenced by the use of the plus-maze, an apparatus that requires animals to turn around at the end of the arms and could

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lead to falling off the maze by larger animals. In addition, the increase in movement by adolescent animals in the plus maze and the corresponding reduction in this effect following CIE exposure is difficult to understand without a corresponding anxiety-like phenotype. Future

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studies investigating CIE in various ages should use less movement intensive tasks such as the zero maze or the light dark box to understand the impact of CIE on anxiety in aged rats. Finally,

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it is important to recall that the behavioral measures where tested in a serial order. While this experimental design allowed for a greater number of measures to be collected, it is possible that the order of tests impacted later data. Additional research should investigate the impact of these measures at different time points to ensure the overall validity of the data. The average age of the population in the United States and the world is continuing to increase. Research has demonstrated this age demographic consumes ethanol, often at levels

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sufficient to produce an ethanol use disorder. It is therefore critical to understand the health implications of ethanol use in the aged population. The current work highlights health risks and specifically risks related to general wellbeing as measured by body weight and overall sensitivity

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to the effects of ethanol. In addition, the current work suggests ethanol tolerance in this age group may develop following moderate chronic exposure. Additional research is needed to

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determine the mechanisms of these effects and fully understand the health implications.

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Van Skike, C. E., Botta, P., Chin, V. S., Tokunaga, S., McDaniel, J. M., Venard, J., . . . Matthews, D. B. (2010). Behavioral effects of ethanol in cerebellum are age dependent: potential system and molecular mechanisms. Alcohol Clin Exp Res, 34(12), 2070-2080. doi:10.1111/j.1530-0277.2010.01303.x Van Skike, C. E., Diaz-Granados, J. L., & Matthews, D. B. (2015). Chronic Intermittent Ethanol Exposure Produces Persistent Anxiety in Adolescent and Adult Rats. AlcoholismClinical and Experimental Research, 39(2), 262-271. doi:10.1111/acer.12617 Varlinskaya, E. I., & Spear, L. P. (2004). Acute ethanol withdrawal (hangover) and social behavior in adolescent and adult male and female Sprague-Dawley rats. Alcohol Clin Exp Res, 28(1), 40-50. doi:10.1097/01.ALC.0000108655.51087.DF Verendeev, A., & Riley, A. L. (2012). Conditioned taste aversion and drugs of abuse: history and interpretation. Neurosci Biobehav Rev, 36(10), 2193-2205. doi:10.1016/j.neubiorev.2012.08.004 Walf, A. A., & Frye, C. A. (2007). The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nature Protocols, 2(2), 322-328. doi:10.1038/nprot.2007.44 Wood, W. G., & Armbrecht, H. J. (1982). Age-Differences in Ethanol-Induced Hypothermia and Impairment in Mice. Neurobiology of Aging, 3(3), 243-246. doi:Doi 10.1016/0197-4580(82)90046-X Wood, W. G., Armbrecht, H. J., & Wise, R. W. (1982). Ethanol intoxication and withdrawal among three age groups of C57BL/6NNIA mice. Pharmacol Biochem Behav, 17(5), 1037-1041. Yankelevitch-Yahav, R., Franko, M., Huly, A., & Doron, R. (2015). The forced swim test as a model of depressive-like behavior. J Vis Exp(97). doi:10.3791/52587

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Figure Captions Figure 1: Experimental Timeline.

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Figure 2: Chronic intermittent alcohol exposure significantly altered the body weight of subjects dependent on age. Adolescent animals put on significantly more body weight than either adult or aged rats when administered saline (top). The effect of low dose alcohol, 1.0 g/kg, reduced the body weight gain of aged rats (middle) while this effect was increased if 2.0 g/kg ethanol was administered (bottom). Error bars denote standard error of the mean.

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Figure 3: Chronic intermittent alcohol exposure differentially impacted body weight due to the age of the subject. Body weight data of subjects during the chronic intermittent treatment period collapsed across time. Adolescent animals administered saline gained significantly more weight compared to adult or aged animals (top) while aged animals administered 1.0 g/kg or 2.0 g/kg alcohol lost weight compared to both the adolescent and aged rats. Error bars denote standard error of the mean, * p < 0.05 compared to adolescent, 1 p < 0.05 compared to adult.

Figure 4: Aged animals are extremely sensitive to the effects of a high dose of alcohol. Average amount of sleep time following a 3.0 g/kg ethanol injection. Error bars denote standard error of the mean. Insert, a main effect of age was found demonstrating aged rats slept significantly longer following the ethanol injection, * p < 0.05.

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Figure 5: Age of the animal does not produce a systematic effect on blood ethanol levels. Average blood alcohol levels 30-min after alcohol injection on the first day of CIE treatment at the three ages.

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Table 1: Average data for various measures. Data are provided as mean with standard error of the mean given in parenthesis. Body weight on CIE1 is the average body weight on the first chronic intermittent ethanol exposure day while Body weight on CIE10 is the average body weight on the last chronic intermittent ethanol exposure day.
in this column indicates body weight of that condition being significantly different from the average body weight of the other two conditions for that age. Open arm entries, %open arm entries, Closed arm entries and Total movement relate to data from the elevated plus maze. * in the Closed arm entries and Total movement columns indicate significantly different from saline in the adolescent age. Immobile bins FST refers to the number of 5-sec times bins scored as immobile in the forced swim test.
in the aged categories indicates a main effect where aged rats are significantly different from adult and adolescent rats. Table 2: Average data for the non-spatial and spatial tasks. Data are provided as mean with standard error of the mean given in parenthesis.  in the non-spatial task indicate adolescent animals on day 1 performing significantly better than adult or aged animals on day 1.  in the non-spatial task indicated aged animals on day 2 and day 3 performing worse than adolescent

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or adult animals on day 2 and day 3.  in the spatial task indicated adolescent animals performing better than adult and aged animals on day 3.

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TABLE 1

25(5.0)

1.0 g/kg ethanol 108.0(2.4)

229.4(2.1)

0.75(.30)

20(7.0)

2.0 g/kg ethanol 108.1(2.5)

222.1(3.7)

0.90(.29)

Saline control

310.1(2.4)  0.00(.00)

104.4(2.5)

6.0(1.4)

3.80(.74)

33.7(2.9)

1.75(.21)*

5.6(3.4)

2.50(.47)*

28.6(2.8)

1.75(.21)*

9.8(3.5)

2.75(.48)*

32.8(1.7)

00(0.00)

1.13(.13)

0.3(0.3)

1.12(.12)

36.7(1.4)

348.0(6.4)

1.06(.26)

30(8.0)

2.19(.25)

9.4(3.4)

3.20(.35)

36.1(1.7)

2.0 g/kg ethanol 320.1(5.0)

336.2(10.1)

0.70(.43)

16(10.0)

2.2(.41)

6.0(3.8)

2.90(.76)

36.7(3.5)

Saline control

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1.0 g/kg ethanol 318.8(2.0)

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313.3(2.6)

Immobile bins FST

24(6.0)

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Adult

Total Movement (arm entry)

2.7(.47)

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175.8(3.5)  1.10(.31)

Saline control

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Adolescent

Percent Open Arm Time

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Body weight Body weight Open Arm %Open Arm Closed Arm CIE1 (grams) CIE10 (grams) Entries Entries Entries

Aged

574.0(14.1) 540.4(11.3)

0.30(.15)

11(5.0)

1.45(.17)

2.1(1.3)

1.75(.28)

36.5(2.5)


1.0 g/kg ethanol 590.6(13.3) 562.7(11.6)

0.67(.24)

25(9.0)

1.58(.20)

11.0(4.3)

2.25(.38)

34.5(4.6)


2.0 g/kg ethanol 575.6(13.9) 504.1(21.2) 0.50(.29)

19(9.0)

1.29(.18)

12.1(7.0)

1.78(.40)

41.5(2.9)


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Non Spatial

Spatial Latency(s)

Pathlength(m)

Latency(s)

Pathlength(m)

Day 1

24.8(1.73)


7.06(0.49)


32.7(1.43)

9.92(0.46)

Day 2

12.5(1.23)

3.46(0.37)

27.8(1.38)

8.55(0.53)

Day 3

10.0(0.79)

2.64(0.24)

21.6(1.99)


6.20(0.67)


Day 1

32.9(2.95)

8.98(0.81)

Day 2

17.6(2.04)

4.81(0.58)

28.0(1.46)

8.26(0.64)

Day 3

10.5(1.73)

2.86(0.50)

31.8(2.06)

9.47(0.83)

Day 1

38.2(1.29)

10.5(0.53)

36.4(2.03)

9.82(0.60)

Day 2

33.3(2.46) 9.05(0.72)

30.1(1.67)

8.09(0.51)

Day 3

24.7(2.3)

30.8(1.98)

8.32(0.58)

AC C

EP

TE D

Aged

SC

32.9(1.79)

M AN U

Adult

RI PT

Adolescent

6.92(0.68)

9.82(0.65)

Chronic Intermittent Treatment

Elevated Plus Maze

Forced Swim Test

Loss of Righting Reflex

49-56 days post CIE

Non-Spatial Learning

EP

TE D

M AN U

SC

Blood ethanol determined CIE 1

AC C

Arrival In Colony

8 daysACCEPTED 21MANUSCRIPT days 42 days post CIE post CIE post CIE

1 day post CIE

RI PT

2 days

Spatial Learning

ACCEPTED MANUSCRIPT

240

Saline

200 180

RI PT

Adolescent Adult Aged

160 140 120 100 80 60 2

4

6

8

10

12

240

1 g/kg

200 180 160

120 100 80

18

20

2

4

6

8

10

12

14

16

18

20

EP

0

220

2 g/kg

AC C

200

Body Weight (% Change)

16

140

TE D

Body Weight (% Change)

220

14

M AN U

0

SC

Body Weight (% Change)

220

180 160 140 120 100

80 60 0

2

4

6

8

10

Test Days

12

14

16

18

20

ACCEPTED MANUSCRIPT

180

Saline

120 100

*

*

Adult

Aged

80 60 40 20 Adolescent 180

1 g/kg

140

*

120 100 80

*1

60 40 20 0 180 160

AC C

140

Adult

EP

Adolescent

TE D

Body Weight (% Change)

160

Body Weight (% Change)

M AN U

0

120

*

100

Aged

2 g/kg

*1

80 60 40 20

0 Adolescent

Adult

Group

RI PT

140

SC

Body Weight (% Change)

160

Aged

AC C EP TE D

60

Aged

40

0

CIE Treatment Condition

RI PT

20

SC

A g ed

A d ul t

A d o le sc en t

LORR (min) 60

M AN U

g/ kg

g/ kg

ro l

80

2. 0

1. 0

co nt

Loss of Righting Reflex (min)

ACCEPTED MANUSCRIPT

*

40

20

0

Age

Adolescent

Adult

AC C EP TE D

0

0 0

2.

1. 2.

g/ k

g/ k

g/ k

g/ k

g/ k

g/ k

g

g

g

g

g

g

0

CIE Treatment Condition

M AN U

0

0

2.

1.

0

1.

Blood Ethanol Concentration (mg/dl) 100

SC

300

Adult

200

Aged

RI PT

ACCEPTED MANUSCRIPT

Adolescent

ACCEPTED MANUSCRIPT

HIGHLIGHTS

AC C

EP

TE D

M AN U

SC

RI PT

1. Aged rats are more impaired by ethanol compared to adult or adolescent rats. 2. Body weights of aged rats is compromised by ethanol. 3. Low dose ethanol does not impact all behaviors in aged rats.