Accepted Manuscript The Effects of Acute Alcohol on Motor Impairments in Adolescent, Adult, and Aged Rats Laura C. Ornelas, Adelle Novier, Candice E. Van Skike, Jaime L. Diaz-Granados, Douglas B. Matthews PII:
S0741-8329(14)20208-4
DOI:
10.1016/j.alcohol.2014.12.002
Reference:
ALC 6480
To appear in:
Alcohol
Received Date: 10 October 2014 Revised Date:
1 December 2014
Accepted Date: 5 December 2014
Please cite this article as: Ornelas L.C., Novier A., Van Skike C.E., Diaz-Granados J.L. & Matthews D.B., The Effects of Acute Alcohol on Motor Impairments in Adolescent, Adult, and Aged Rats, Alcohol (2015), doi: 10.1016/j.alcohol.2014.12.002. 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.
ACCEPTED MANUSCRIPT 1
RI PT
The Effects of Acute Alcohol on Motor Impairments in Adolescent, Adult, and Aged Rats
Laura C. Ornelas1, Adelle Novier1, Candice E. Van Skike1, Jaime L. Diaz-Granados1, Douglas B. Matthews1,2
Baylor University, One Bear Place #97334, Waco, Texas 76798, USA
2
University of Wisconsin – Eau Claire, HHH273, Eau Claire, WI 54702, USA
AC C
EP
TE D
Please direct all correspondence to: Douglas B. Matthews, PhD University of Wisconsin – Eau Claire Department of Psychology Eau Claire, WI 54702 Telephone: +1 715 836 2119 Fax: +1 715 836 2124 Email: [email protected]
M AN U
SC
1
ACCEPTED MANUSCRIPT 2
Abstract Acute alcohol exposure has been shown to produce differential motor impairments between aged and adult rats and between adolescent and adult rats. However, the effects of acute
RI PT
alcohol exposure among adolescent, adult, and aged rats have yet to be systematically investigated within the same project using a dose-dependent analysis. We sought to determine the age- and dose-dependent effects of acute alcohol exposure on gross and coordinated motor
SC
performance across the rodent lifespan. Adolescent (PD 30), adult (PD 70), and aged
(approximately 18 months) male Sprague-Dawley rats were tested on 3 separate motor tasks:
M AN U
aerial righting reflex (ARR), accelerating rotarod (RR), and loss of righting reflex (LORR). In a separate group of animals, blood ethanol concentrations (BEC) were determined at multiple time points following a 3.0 g/kg ethanol injection. Behavioral tests were conducted with a Latin square repeated-measures design in which all animals received the following doses: 1.0 g/kg or
TE D
2.0 g/kg alcohol or saline over 3 separate sessions via intraperitoneal (i.p.) injection. During testing, motor impairments were assessed on the RR 10 min post-injection and on ARR 20 min post-injection. Aged animals spent significantly less time on the RR when administered 1.0 g/kg
EP
alcohol compared to adult rats. In addition, motor performance impairments significantly increased with age after 2.0 g/kg alcohol administration. On the ARR test, aged rats were more
AC C
sensitive to the effects of 1.0 g/kg and 2.0 g/kg alcohol compared to adolescents and adults. Seven days after the last testing session, animals were given 3.0 g/kg alcohol and LORR was examined. During LORR, aged animals slept longer compared to adult and adolescent rats. This effect cannot be explained solely by BEC levels in aged rats. The present study suggests that acute alcohol exposure produces greater motor impairments in older rats when compared to adolescent and adult rats and begins to establish a procedure to determine motor effects by alcohol across the lifespan.
ACCEPTED MANUSCRIPT 3
Highlights: Acute alcohol impairs motor performance more in aged rats than in younger rats.
•
Aged animals are more sensitive to the hypnotic effects of alcohol.
•
Aerial righting reflex is a more reliable measure of motor impairments than rotarod.
RI PT
•
AC C
EP
TE D
M AN U
SC
Keywords: alcohol, aerial righting reflex, rotarod, aging, loss of righting reflex
ACCEPTED MANUSCRIPT 4
The average age of the world’s population is rapidly rising (Lutz, Sanderson, & Scherbov, 2008). According to the United States Census Bureau, individuals aged 65 and older are projected to total approximately 72 million people and represent nearly 20% of the total U.S.
RI PT
population in the year 2030 (He, Sengupta, Velkoff, & DeBarros, 2005). Importantly,
approximately 50% of the elderly population (aged over 65) and almost 25% of individuals over 85 years old currently drink alcohol (Caputo et al., 2012). In addition, nearly 13% of men and
SC
8% of women over the age of 65 consume alcohol in a binge-drinking manner (Blazer & Wu, 2009). In the United States, it is estimated that the prevalence of alcohol-use disorders among the
M AN U
elderly population is approximately 1–3% (Caputo et al., 2012). As the global population continues to increase, substantial alcohol consumption and its consequences in the elderly are an important, but understudied, public health concern (Babor, 2010).
Individuals aged 65 years and older are especially susceptible to the risk factors
TE D
associated with alcohol consumption. Increased alcohol consumption in aged individuals could be a contributing factor to cognitive deficits including dementia (Thomas & Rockwood, 2001), and to impaired motor coordination and increased falls in older individuals (Høidrup, Grønbæk,
EP
Gottschau, Lauritzen, & Schroll, 1999; Jones, Cyr, & Patil, 1994; Mukamal et al., 2004; Mukamal, Robbins, Cauley, Kern, & Siscovick, 2007; Weafer & Fillmore, 2012). Deficits in
AC C
motor coordination in this age group may be related to age-dependent changes in cerebellar function (Piguet et al., 2006). Alcohol has been shown to decrease cell density and size of the cerebellar vermis, resulting in gait ataxia (Johnson-Greene et al., 1997; Phillips, Harper, & Kril, 1990; Piguet et al., 2006). Therefore, the aging cerebellum paired with heavy alcohol consumption could lead to increased motor impairments in the elderly.
ACCEPTED MANUSCRIPT 5
In concordance with age-related effects of alcohol in humans, research using animal models has shown that many of the effects of alcohol are age-dependent (Chin, Van Skike, & Matthews, 2010 for review). Adolescent rats, compared to adult rats, have been shown to be less
RI PT
sensitive to the sedative (Little, Kuhn, Wilson, & Swartzwelder, 1996), anxiogenic (Doremus, Brunell, Varlinskaya, & Spear, 2003), hypnotic (Matthews, Tinsley, Diaz-Granados, Tokunaga, & Silvers, 2008; Silveri & Spear, 1998), and motor impairing effects of acute alcohol (Ramirez
SC
& Spear, 2010; Van Skike et al., 2010; White et al., 2002). Furthermore, adolescent rodents exhibit significantly more acute tolerance compared to adult rodents (Draski, Bice, & Deitrich,
M AN U
2001; Grieve & Littleton, 1979; Silveri & Spear, 1998, 2002; Varlinskaya & Spear, 2006) and are more sensitive to alcohol-induced hypothermia (Ristuccia & Spear, 2008). Research indicates that older rodents are more sensitive to acute alcohol exposure compared to adolescent and adult rats (Novier, Van Skike, Diaz-Granados, Mittleman, &
TE D
Matthews, 2013; Van Skike et al., 2010). Older rodents are more sensitive to the hypnotic (Ott, Hunter, & Walker, 1985) and hypothermic effects of acute alcohol (Wood & Armbrecht, 1982), as well as the severity of alcohol withdrawal (Wood, Armbrecht, & Wise, 1982) compared to
EP
younger rodents. To explore the effects of alcohol across the lifespan, Van Skike et al. (2010) examined the impact of a single acute alcohol dose on motor impairments in 4 rodent age groups.
AC C
Aged rats showed significantly more impairment in motor performance compared to periadolescent and adolescent rats. Furthermore, Novier et al. (2013) investigated the differences in the motor and memory-impairing effects of acute alcohol between adult and aged rats. Similarly, results indicate that aged animals performed significantly worse in all behavioral measures compared to adult rats.
ACCEPTED MANUSCRIPT 6
Although current research has recognized differential motor impairments between adolescent and aged rats (Van Skike et al., 2010; White et al., 2002) and adult and aged rats (Novier et al., 2013), research has yet to systematically investigate the effect of acute ethanol on
RI PT
motor impairments across the lifespan using a dose-dependent analysis. In addition, the effect of age on a high dose of alcohol-induced hypnosis has yet to be investigated. Therefore, we sought to determine the age- and dose-dependent effects of acute alcohol exposure on gross and
SC
coordinated motor performance in adolescent, adult, and aged rats using the accelerated rotarod (RR) and aerial righting reflex (ARR). In addition, the effect of 3.0 g/kg on ethanol-induced loss
M AN U
of righting reflex (LORR) was determined. Finally, blood ethanol concentrations (BEC) were determined in a separate group of animals at 7 different time points following a 3.0 g/kg ethanol injection to better understand how BEC affects LORR at 3 different ages. We present evidence that alcohol produces greater motor impairments in older rats when compared to adolescent and
concentrations.
Subjects
EP
Materials and methods
TE D
adult rats and these motor impairments are not completely explained by blood ethanol
Twelve adolescent (postnatal day (PD) 28), 12 young adult (approximately PD 70), and
AC C
12 aged (approximately 18 months) male Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). These ages were selected because PD 28–30 is a developmental period of early adolescence based on evidence that mature sperm is not yet found, while all sperm are mature at PD 70, indicating this age is the beginning of adulthood (Odell, 1990). Aged animals were 18 months, to be consistent with our previous work (Novier et al., 2013). Animal care procedures were approved by the Institutional Animal Care and Use Committee of Baylor University. Animals were individually housed and given ad libitum access to food and water
ACCEPTED MANUSCRIPT 7
throughout the experiment. Following previously published methods, all rats acclimated to the colony room for 2 days before any experimental procedures (Chin et al., 2011; Novier, Van Skike, Chin, Diaz-Granados, & Matthews, 2012; Novier et al., 2013; Silvers et al., 2006;
RI PT
Tokunaga, Silvers, & Matthews, 2006; Van Skike, Novier, Diaz-Granados, & Matthews, 2012). To investigate the dose-dependent effects of acute ethanol exposure in the same animals and therefore reduce subject number, animals were involved in a Latin Square repeated-measures
SC
design with experimental doses of 1.0 g/kg or 2.0 g/kg (10% w/v) alcohol, or a saline dose
equivalent to the volume of a 1.0 g/kg dose of alcohol. Animals were first tested on the RR at
M AN U
10 min post ethanol or saline injection, and ARR was assessed 20 min post ethanol or saline injection. Three separate trials were conducted 3 days apart to minimize carryover effects. The first test session occurred 24 h after the last RR training trial (see below). Animals were randomly assigned to each of the 3 different drug orders and counterbalanced by age and dose
TE D
for all 3 trials. Seven days after the last testing trial, a subset of animals (n = 4 per age group) were given an acute injection of 3.0 g/kg alcohol and loss of righting (LORR) was determined. Finally, a separate group of adolescent, adult, and aged animals (n = 6 per age) were injected i.p.
EP
with 3.0 g/kg ethanol. The tail was nicked and blood was collected for BEC analysis via the Analox AM1 protocol 30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min post-
AC C
injection.
Aerial righting reflex (ARR)
The effect of acute alcohol exposure on gross motor impairment was assessed 20 min
after saline or alcohol administration by ARR as previously described (Novier et al., 2013; Van Skike et al., 2010). The 3 test trials occurred on PD 31, PD 35, and PD 39 in adolescent rats, and on PD 73, PD 77, and PD 81 in adult rats. Aged animals are classified as approximately
ACCEPTED MANUSCRIPT 8
18 months old by the supplier and therefore exact ages are unknown. A ruler was vertically taped above a 10-inch foam pad. Animals were initially released 5 inches (12.7 cm) above the foam pad in a supine position. An animal’s righting reflex was considered successful if 3 out of 4 paws
RI PT
made direct contact with the foam pad on 2 out of 3 releases. If righting was not successful, the height of release was increased in 5-inch (12.7 cm) increments up to a maximum height of 25 inches (63.5 cm). Subjects who failed to achieve successful righting reflex at 25 inches
SC
(63.5 cm) were given a score of 30 inches (76.2 cm) for statistical analysis. Accelerating rotarod (RR)
M AN U
Apparatus
RR was used to investigate the effects of alcohol exposure on motor coordination. Motor activity was tested on a 4-station Rota-Rod treadmill (Model ENV 575, Med Associates, St. Albans, VT). The apparatus was located in a behavioral room isolated from animal caging
Training
TE D
and housing.
Subjects received 5 consecutive training trials on the RR, as previously described (Novier
EP
et al., 2013). Adolescents received RR training on PD 30 and adults on PD 72. The rod accelerated from 4 rpm to 40 rpm over a 5-min period. The rotarod is covered with fine grit
AC C
sandpaper to provide a uniform surface and to reduce slipping (Rustay, Wahlsten, & Crabbe, 2003). The RR was interfaced to a computer that collected the time each subject remained on the rod up to a maximum time of 360 sec. After the 5 training trials were complete, averages of the last 3 trials were calculated and animals who failed to meet a criterion of 7 sec were given additional training trials until their final 3 training trials averaged 7 sec. Testing
ACCEPTED MANUSCRIPT 9
Subjects received an i.p. injection of 1.0 g/kg alcohol, 2.0 g/kg alcohol, or a saline equivalent dose. Motor activity on the RR was recorded 10 min after alcohol or saline administration. The 3 test trials occurred on PD 31, PD 35, and PD 39 in adolescent rats, and on
RI PT
PD 73, PD 77, and PD 81 in adult rats. Aged animals are classified as approximately 18 months old by the supplier and therefore exact ages are unknown. Loss of Righting Reflex (LORR)
SC
Procedure
The sedative/hypnotic effect of alcohol was assessed using the loss of righting reflex
M AN U
paradigm. One week after the last testing trial (PD 46 for adolescents and PD 88 for adults), a subset of previously tested animals (n = 4) received an i.p. injection of 3.0 g/kg alcohol. Animals were monitored throughout LORR. Latency to regain righting reflex was assessed. Recovery of reflex was defined as successfully righting 3 times in 1 min.
TE D
Blood ethanol concentration Procedure
To better understand how age impacts blood ethanol levels, 6 adolescent rats, 6 adult rats,
EP
and 6 aged rats were injected with 3.0 g/kg ethanol i.p. before collection of blood via the tail. Animals were the same age as those previously assessed for LORR to allow for a comparison of
AC C
how BEC influences righting reflex. Following injection, the tail was nicked and blood collected at 7 different time points (30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min) and BEC was assessed via the AM1 Analox system following manufacturer’s recommendations. Results Aerial Righting Reflex A two-way repeated measure ANOVA revealed a significant Age × Dose interaction, F(4,15) = 6.79, p < 0.05. To further investigate the significant interaction, one-way ANOVAs for
ACCEPTED MANUSCRIPT 10
age were performed at each of the 3 doses tested. Importantly, no significant differences by age were found when subjects were tested with saline, F(2,15) = 1.00, p = 0.39, indicating no baseline or carry over effects in performance (see Fig. 1A). A significant effect was found when
RI PT
animals were tested with 1.0 g/kg alcohol, F(2,15) = 9.80, p < 0.05. Bonferroni post hoc tests revealed that aged rats were more sensitive to the effects of 1.0 g/kg alcohol compared to adolescents, t = 3.84, p < 0.05, and adults, t = 3.84, p < 0.05 (see Fig. 1B). Similarly, a
SC
significant effect was found when rats were tested with 2.0 g/kg alcohol, F(2,15) = 10.19,
p < 0.05. Specifically, aged animals performed worse on ARR compared to adolescent, t = 4.37,
M AN U
p < 0.05, and adult rats, t = 3.17, p < 0.05 following the 2.0 g/kg dose (see Fig. 1C). Finally, adolescent and adult rats did not significantly differ in ARR performance when given 1.0 g/kg alcohol, t = 0.00, p > 0.05 or 2.0 g/kg alcohol, t = 1.20, p > 0.05. Accelerating rotarod
TE D
There was a significant main effect of age during RR training, F(2,15) = 7.57, p < 0.05. Bonferroni post hoc tests revealed a significant difference between adolescent and aged rats, t = 3.50, p < 0.05, and between adolescent and adult rats in training performance, t = 3.22,
EP
p < 0.05 (see Table 1). However, adult and aged animals did not significantly differ from one another on RR training performance, t = 0.29, p > 0.05. To equate for differential baseline
AC C
performance, the effect of acute alcohol on RR performance was analyzed by percent change from each animal’s baseline performance (see Table 1 for untransformed data). A two-way repeated measures ANOVA revealed a significant Age × Dose interaction
during RR testing, F(4,15) = 11.76, p < 0.05. One-way ANOVAs were performed to assess the significant differences between each individual dose tested. A significant effect was found when animals were tested with 1.0 g/kg alcohol, F(2,15) = 6.51, p < 0.05. Bonferroni post hoc tests
ACCEPTED MANUSCRIPT 11
showed aged animals performed worse on RR compared to adult rats when administered 1.0 g/kg alcohol, t = 3.46, p < 0.05 (see Fig. 2A). A significant effect was found when animals were tested with 2.0 g/kg alcohol, indicating motor performance impairments increased with age,
RI PT
F(2,15) = 4.28, p < 0.05 (however, post hoc test revealed no significant effects between ages) (see Fig. 2B). A one-way ANOVA revealed no significant differences between ages when tested
SC
with saline, F(2,15) = 2.80, p > 0.05. Loss of righting reflex
A one-way ANOVA revealed a significant difference in sleep time, F(2,9) = 8.02,
M AN U
p < 0.05. Bonferroni post hoc analysis showed aged animals slept longer (mean sleep time of 157.0 min and a standard error of the mean of 53.0 min) compared to adult (mean sleep time of 0 min and a standard error of the mean of 0 min), t = 3.56, p < 0.05, and adolescent rats (mean sleep time of 8.25 min and a standard error of the mean of 8 min), t = 3.37, p < 0.05. No
p > 0.05.
TE D
significant difference in sleep time was found between adolescent and adult rats, t = 0.19,
Blood ethanol concentration
EP
A two-way ANOVA revealed a significant Age × Time interaction on blood ethanol
AC C
concentration, F(12,90) = 23.41, p < 0.001. One-way ANOVAs were performed to assess the significant differences at each time tested. Adolescent blood ethanol levels were significantly greater 30 min post-injection compared to both adult and aged animals (Tukey post hoc comparisons, both p's < 0.05). However, adolescent blood ethanol concentrations were significantly less than adults (p < 0.05) and aged animals (p < 0.001) at the 240-, 300-, and 360-min time points. Finally, adult blood ethanol concentrations were significantly different from aged animals 360 min post-ethanol injection (p < 0.001) (see Fig. 3).
ACCEPTED MANUSCRIPT 12
Discussion The current study examined the age-and dose-dependent effects of acute alcohol exposure on gross and coordinated motor performance across the rodent lifespan and provides a
RI PT
partial ethanol clearance curve following a high-dose ethanol administration. On the ARR and RR tasks, aged rats exhibited greater deficits in motor performance compared to both adolescent and adult rats during acute alcohol exposure at both doses. In addition, aged animals slept longer
SC
compared to both adult and adolescent rats in the LORR task. To our knowledge, this study is the first to report a difference in motor impairments and sedative/hypnotic effects of acute alcohol
following a high-dose ethanol exposure.
M AN U
among adolescent, adult, and aged rats and provides novel data concerning blood ethanol levels
Research from our laboratory has investigated the age-dependent effects of acute alcohol exposure on motor impairments (Novier et al., 2013; Van Skike et al., 2010). The results of the
TE D
present study are in accordance with previous research that shows age-dependent impairments in ARR performance after acute alcohol administration (Van Skike et al., 2010). However, Van Skike et al. (2010) reported adult and aged rats did not significantly differ from each other
EP
during the ARR paradigm, whereas in the present set of studies aged animals were significantly more impaired relative to adults. A possible explanation for this discrepancy is our adult rats
AC C
were tested between PND 73 and PND 88, while Van Skike et al. (2010) tested adult rats at PD 120. In addition, Van Skike et al. (2010) assessed gross motor performance using only the ARR task. The current study included the RR as an additional measure of motor skill learning to investigate the age-dependent differences in motor performance associated with acute alcohol exposure prior to the ARR task. It is possible that the sequential nature of the testing procedure in the current set of studies altered ARR performance and may have produced greater ARR
ACCEPTED MANUSCRIPT 13
deficits in aged subjects in the current experiments. Future research using multiple measures of behavior within a single ethanol dose needs to address potential sequential effects. Novier et al. (2013) found that aged animals showed greater alcohol-induced ataxia on
RI PT
the RR compared to adult rats when animals were tested with moderate to high alcohol doses. However, Novier et al. (2012) found that adolescent and adult animals had facilitated RR performance following administration of a low (1.0 g/kg) dose of alcohol, a result that is
SC
replicated for adult animals but not adolescent animals in the current work. Furthermore, the current work is the first to investigate both low (1.0 g/kg) and moderately high (2.0 g/kg) doses
M AN U
of ethanol on the rotarod across a full age range. The current work extends previous findings to demonstrate that aged animals are still impaired on the rotarod at a low dose of alcohol and this impairment is magnified as the dose of ethanol increases. Additionally, in agreement with results from the present study, Novier et al. (2013) reported no significant differences between adult and
TE D
aged rats in RR training performance. However, the experimental procedures for RR training were slightly different compared to our previous studies (Novier et al., 2013). Due to a small sample size, animals who failed to meet criterion at 7 sec in the present study were given
EP
additional training trials until their final 3 training trials averaged 7 sec, rather than being excluded from RR testing as was done previously. Such a training procedure may bias our results
AC C
and confound a potential impairment due to a floor effect. Future studies should use a slower acceleration speed or a fixed speed task to determine if aged animals are indeed more impaired by acute ethanol on this task. Finally, aged animals slept longer compared to adolescent and adult rats during LORR.
These results are consistent with previous studies, which state that adolescent animals are less sensitive to alcohol-induced sedative/hypnotic effects (Little et al., 1996; Matthews et al., 2008;
ACCEPTED MANUSCRIPT 14
Silveri & Spear, 1999). While BECs were not taken from animals when they regained their righting reflex, we did investigate BEC in a separate group of same-aged adolescent, adult, and aged animals following a 3.0 g/kg ethanol injection. Initially, adolescent animals demonstrate
RI PT
higher BEC than either adult or aged animals, but adolescents have lower BEC compared to the other 2 age groups following the 240-min time point until the last blood collection time. Such a time-dependent interaction of age and BEC is intriguing and suggests that behaviors impacted by
SC
alcohol may also show a time-dependent effect when different ages are tested at different times in the BEC curve. Furthermore, the BEC data provide some important insight into the LORR
M AN U
data as it relates to the question of whether aged animals are more sensitive to a high dose of alcohol than adolescent or adult animals. Specifically, aged rats slept approximately 150 min following administration of 3.0 g/kg ethanol, a time point when the BEC clearance study suggests blood ethanol levels among the 3 ages should be similar (no significant difference in
TE D
BEC at either the 120-min or 180-min time point between adolescent, adult, and aged rats). However, without determining BEC in subjects undergoing the LORR experiment, future research will need to determine if aged animals are indeed more sensitive to acute ethanol on the
EP
LORR test. Furthermore, BEC in aged animals remained high throughout the experiment even though collection times occurred 6 h after injection. This suggests that the aged system is
AC C
impaired in clearance of the drug and future studies should address this issue across a full ethanol dose range. In addition, there is a lack of ethanol clearance in the adult animals until the final time point. Such a lack of clearance was unpredicted and highlights the potential limitation of using i.p. injections to study the effects of ethanol and the need to collect BEC levels from animals used in future LORR studies. Finally, the impact of sex and age on ethanol’s effects is
ACCEPTED MANUSCRIPT 15
understudied, particularly for aged animals. Future studies should include female subjects to better understand how sex and age interact. The continuous decline of motor function is a widespread feature of aging (Desrosiers,
RI PT
Hébert, Bravo, & Dutil, 1995; Ranganathan, Siemionow, Sahgal, & Yue, 2001; Smith et al., 1999). Age-related deficits in motor function may be the consequence of age-related muscle deterioration, or sarcopenia (Herndon et al., 2002), leading to impaired mobility, gait
SC
abnormalities, and increased risk of falls (Hunter, White, & Thompson, 1998). Previous research has indicated that aged rats have deficits in motor coordination, balance, and reflexes as
M AN U
indicated by the RR and other tasks of motor performance (Shukitt-Hale, Mouzakis, & Joseph, 1998; Wallace, Krauter, & Campbell, 1980). These deficits may be associated with agedependent decline in strength and muscle mass (Hepple, Ross, & Rempfer, 2004). In addition, alcohol metabolism changes with age, which may result in aged humans being more sensitive to
TE D
the toxic effects of alcohol (Seitz et al., 1993). Seitz et al. (1993) reported gastric alcohol dehydrogenase (ADH) activity was significantly lower in elderly male subjects. Decreased ADH may contribute to reduced first pass metabolism, leading to an increase in blood alcohol
EP
concentrations as well as impairments in motor coordination (Seitz et al., 1993). This raises the possibility that aged animals in the current project are more impaired by acute alcohol due to
AC C
differential BECs (see Fig. 3). Although we did not measure BEC following the lower injection amounts of 1.0 g/kg and 2.0 g/kg ethanol, we do not think differential BEC levels explain the current data for two reasons. First, alcohol was administered i.p. and therefore different ADH activity in the gut would not alter BEC levels. Second, previous work from our laboratory has shown, using identical ages and the same lower alcohol doses, that while alcohol produces greater cognitive and motor impairment in aged animals compared to adult animals, BEC did not
ACCEPTED MANUSCRIPT 16
predict greater alcohol impairment in aged animals (Novier et al., 2013). However, when feasible, future projects should continue to monitor BEC in studies investigating the effect of alcohol across different ages.
RI PT
The present study provides evidence that the ARR paradigm appears to be a better
assessment of motor performance compared to the RR task. Animals’ performance on the ARR task was consistent throughout each testing trial and provided less variability in motor
SC
performance compared to RR. In addition, there is no significant baseline difference. A possible explanation as to why the RR produced differences in motor impairments compared to the ARR
M AN U
could be the specific design of the RR. The size of the cylindrical rotating rod was the same size in diameter for all 3 age groups tested. In addition, the acceleration of the rod might not be the best procedure to test performance in this task. Future studies that use the RR to assess motor performance on aged animals should adjust RR parameters to accommodate changes in body
TE D
weight and size of the animals.
In conclusion, the results of the present study show that acute alcohol administration produces age- and dose-dependent effects on motor performance. With the inclusion of 3
EP
different developmental age groups, the current study provides a more comprehensive view of age-dependent motor impairments during the rodent lifespan. The present study adds to the
AC C
growing body of research that indicates alcohol produces age-dependent impairments in motor performance.
ACCEPTED MANUSCRIPT 17
References
RI PT
Babor, T. (ed.). (2010). Alcohol: No Ordinary Commodity: Research and Public Policy. (Oxford: Oxford University Press).Blazer, D. G., & Wu, L. T. (2009). The epidemiology of at-risk and binge drinking among middle-aged and elderly community adults: National Survey on Drug Use and Health. The American Journal of Psychiatry, 166, 1162–1169.Caputo, F., Vignoli, T., Leggio, L., Addolorato, G., Zoli, G., & Bernardi, M. (2012). Alcohol use disorders in the elderly: a brief overview from epidemiology to treatment options. Experimental Gerontology, 47, 411–416. Chin, V. S., Van Skike, C. E., Berry, R. B., Kirk, R. E., Diaz-Granados, J., & Matthews, D. B. (2011). Effect of acute ethanol and acute allopregnanolone on spatial memory in adolescent and adult rats. Alcohol, 45, 473–483.
SC
Chin, V. S., Van Skike, C. E., & Matthews, D. B. (2010). Effects of ethanol on hippocampal function during adolescence: a look at the past and thoughts on the future. Alcohol, 44, 3– 14.
M AN U
Desrosiers, J., Hébert, R., Bravo, G., & Dutil, É. (1995). Upper extremity performance test for the elderly (TEMPA): normative data and correlates with sensorimotor parameters. Archives of Physical Medicine and Rehabilitation, 76, 1125–1129. Doremus, T. L., Brunell, S. C., Varlinskaya, E. I., & Spear, L. P. (2003). Anxiogenic effects during withdrawal from acute ethanol in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 75, 411–418.
TE D
Draski, L. J., Bice, P. J., & Deitrich, R. A. (2001). Developmental alterations of ethanol sensitivity in selectively bred high and low alcohol sensitive rats. Pharmacology, Biochemistry, and Behavior, 70, 387–396. Grieve, S. J., & Littleton, J. M. (1979). Age and strain differences in the rate of development of functional tolerance to ethanol by mice. The Journal of Pharmacy and Pharmacology, 31, 696–700.
EP
He, W., Sengupta, M., Velkoff, V. A., & DeBarros, K. A. (2005). 65+ in the United States, 2005. (Washington, DC: U.S. Government Printing Office).
AC C
Hepple, R. T., Ross, K. D., & Rempfer, A. B. (2004). Fiber atrophy and hypertrophy in skeletal muscles of late middle-aged Fischer 344 × Brown Norway F1-hybrid rats. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 59, 108–117. Herndon, L. A., Schmeissner, P. J., Dudaronek, J. M., Brown, P. A., Listner, K. M., Sakano, Y., et al. (2002). Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature, 419, 808–814. Høidrup, S., Grønbæk, M., Gottschau, A., Lauritzen, J. B., & Schroll, M. (1999). Alcohol intake, beverage preference, and risk of hip fracture in men and women. Copenhagen Centre for Prospective Population Studies. American Journal of Epidemiology, 149, 993–1001. Hunter, S., White, M., & Thompson, M. (1998). Techniques to evaluate elderly human muscle function: a physiological basis. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 53, B204–B216.
ACCEPTED MANUSCRIPT 18
Johnson-Greene, D., Adams, K. M., Gilman, S., Kluin, K. J., Junck, L., Martorello, S., et al. (1997). Impaired upper limb coordination in alcoholic cerebellar degeneration. Archives of Neurology, 54, 436–439. Jones, T. V., Cyr, D. G., & Patil, K. (1994). Effects of alcohol measured by dynamic posturography in elderly women. Journal of the American Geriatrics Society, 42, Sa72.
RI PT
Little, P. J., Kuhn, C. M., Wilson, W. A., & Swartzwelder, H. S. (1996). Differential effects of ethanol in adolescent and adult rats. Alcoholism: Clinical and Experimental Research, 20, 1346–1351. Lutz, W., Sanderson, W., & Scherbov, S. (2008). The coming acceleration of global population ageing. Nature, 451, 716–719.
SC
Matthews, D. B., Tinsley, K. L., Diaz-Granados, J. L., Tokunaga, S., & Silvers, J. M. (2008). Chronic intermittent exposure to ethanol during adolescence produces tolerance to the hypnotic effects of ethanol in male rats: a dose-dependent analysis. Alcohol, 42, 617–621.
M AN U
Mukamal, K. J., Mittleman, M. A., Longstreth, W. T., Newman, A. B., Fried, L. P., & Siscovick, D. S. (2004). Self‐reported alcohol consumption and falls in older adults: cross‐sectional and longitudinal analyses of the cardiovascular health study. Journal of the American Geriatrics Society, 52, 1174–1179. Mukamal, K. J., Robbins, J. A., Cauley, J. A., Kern, L. M., & Siscovick, D. S. (2007). Alcohol consumption, bone density, and hip fracture among older adults: the cardiovascular health study. Osteoporosis International, 18, 593–602.
TE D
Novier, A., Van Skike, C. E., Chin, V. S., Diaz-Granados, J. L., & Matthews, D. B. (2012). Low and moderate doses of acute ethanol do not impair spatial cognition but facilitate accelerating rotarod performance in adolescent and adult rats. Neuroscience Letters, 512, 38–42.
EP
Novier, A., Van Skike, C. E., Diaz‐Granados, J. L., Mittleman, G., & Matthews, D. B. (2013). Acute alcohol produces ataxia and cognitive impairments in aged animals: a comparison between young adult and aged rats. Alcoholism: Clinical and Experimental Research, 37, 1317–1324.
AC C
Odell, W. D. (1990). Sexual maturation in the rat. In Control of the Onset of Puberty, M. M. Grumback, P. C. Sizonenko, & M. L. Aubert, eds. (Baltimore, MD: Williams and Williams), pp. 183–210. Ott, J. F., Hunter, B. E., & Walker, D. W. (1985). The effect of age on ethanol metabolism and on the hypothermic and hypnotic responses to ethanol in the Fischer 344 rat. Alcoholism: Clinical and Experimental Research, 9, 59–65. Phillips, S. C., Harper, C. G., & Kril, J. J. (1990). The contribution of Wernicke's encephalopathy to alcohol-related cerebellar damage. Drug and Alcohol Review, 9, 53– 60. Piguet, O., Cramsie, J., Bennett, H. P., Kril, J. J., Lye, T. C., Corbett, A. J., et al. (2006). Contributions of age and alcohol consumption to cerebellar integrity, gait and cognition in non-demented very old individuals. European Archives of Psychiatry and Clinical Neuroscience, 256, 504–511.
ACCEPTED MANUSCRIPT 19
Ramirez, R. L., & Spear, L. P. (2010). Ontogeny of ethanol-induced motor impairment following acute ethanol: assessment via the negative geotaxis reflex in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 95, 242–248. Ranganathan, V. K., Siemionow, V., Sahgal, V., & Yue, G. H. (2001). Effects of aging on hand function. Journal of the American Geriatrics Society, 49), 1478–1484.
RI PT
Ristuccia, R. C., & Spear, L. P. (2008). Autonomic responses to ethanol in adolescent and adult rats: a dose–response analysis. Alcohol, 42, 623–629. Rustay, N. R., Wahlsten, D., & Crabbe, J. C. (2003). Influence of task parameters on rotarod performance and sensitivity to ethanol in mice. Behavioural Brain Research, 141, 237– 249.
SC
Seitz, H. K., Egerer, G., Simanowski, U. A., Waldherr, R., Eckey, R., Agarwal, D. P., et al. (1993). Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism. Gut, 34, 1433–1437.
M AN U
Shukitt-Hale, B., Mouzakis, G., & Joseph, J. A. (1998). Psychomotor and spatial memory performance in aging male Fischer 344 rats. Experimental Gerontology, 33, 615–624. Silveri, M. M., & Spear, L. P. (1998). Decreased sensitivity to the hypnotic effects of ethanol early in ontogeny. Alcoholism: Clinical and Experimental Research, 22, 670–676. Silveri, M. M., & Spear, L. P. (1999). Ontogeny of rapid tolerance to the hypnotic effects of ethanol. Alcoholism: Clinical and Experimental Research, 23, 1180–1184.
TE D
Silveri, M. M., & Spear, L. P. (2002). The effects of NMDA and GABAA pharmacological manipulations on ethanol sensitivity in immature and mature animals. Alcoholism: Clinical and Experimental Research, 26, 449–456. Silvers, J. M., Tokunaga, S., Mittleman, G., O'Buckley, T., Morrow, A. L. & Matthews, D. B. (2006). Chronic intermittent ethanol exposure during adolescence reduces the effect of ethanol challenge on hippocampal allopregnanolone levels and Morris water maze task performance. Alcohol, 39, 151–158.
EP
Smith, C. D., Umberger, G. H., Manning, E. L., Slevin, J. T., Wekstein, D. R., Schmitt, F. A., et al. (1999). Critical decline in fine motor hand movements in human aging. Neurology, 53, 1458–1461.
AC C
Thomas, V. S., & Rockwood, K. J. (2001). Alcohol abuse, cognitive impairment, and mortality among older people. Journal of the American Geriatrics Society, 49, 415–420. Tokunaga, S., Silvers, J. M., & Matthews, D. B. (2006). Chronic intermittent ethanol exposure during adolescence blocks ethanol-induced inhibition of spontaneously active hippocampal pyramidal neurons. Alcoholism: Clinical and Experimental Research, 30, 1–6. Van Skike, C. E., Botta, P., Chin, V. S., Tokunaga, S., McDaniel, J. M., Venard, J., et al. (2010). Behavioral effects of ethanol in cerebellum are age dependent: potential system and molecular mechanisms. Alcoholism: Clinical and Experimental Research, 34, 2070– 2080.
ACCEPTED MANUSCRIPT 20
Van Skike, C. E., Novier, A. K., Diaz-Granados, J. L., & Matthews, D. B. (2012). The effect of chronic intermittent ethanol exposure on spatial memory in adolescent rats: The dissociation of metabolic and cognitive tolerance. Brain Research, 1453, 34–39.
RI PT
Varlinskaya, E. I., & Spear, L. P. (2006). Ontogeny of acute tolerance to ethanol‐induced social inhibition in Sprague–Dawley rats. Alcoholism: Clinical and Experimental Research, 30, 1833–1844. Wallace, J. E., Krauter, E. E., & Campbell, B. A. (1980). Motor and reflexive behavior in the aging rat. Journal of Gerontology, 35, 364–370.
SC
Weafer, J., & Fillmore, M. T. (2012). Acute tolerance to alcohol impairment of behavioral and cognitive mechanisms related to driving: drinking and driving on the descending limb. Psychopharmacology (Berl), 220, 697–706. White, A. M., Truesdale, M. C., Bae, J. G., Ahmad, S., Wilson, W. A., Best, P. J., et al. (2002). Differential effects of ethanol on motor coordination in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 73, 673–677.
M AN U
Wood, W. G., & Armbrecht, H. J. (1982). Behavioral effects of ethanol in animals: age differences and age changes. Alcoholism: Clinical and Experimental Research, 6, 3–12.
AC C
EP
TE D
Wood, W. G., Armbrecht, H. J., & Wise, R. W. (1982). Ethanol intoxication and withdrawal among three age groups of C57BL/6NNIA mice. Pharmacology, Biochemistry, and Behavior, 17, 1037–1041.
ACCEPTED MANUSCRIPT 21
Figure captions Figure 1. Aged animals are more sensitive to the gross motor effects of acute ethanol compared to adolescent and adult rats. (A) ARR height achieved after saline (B) ARR height achieved after 1.0 g/kg ethanol (C) ARR height achieved after 2.0 g/kg ethanol, *p < 0.05, error bars denote S.E.M.
RI PT
Figure 2. Coordinated motor impairments on RR increased with age following acute ethanol administration. (A) RR performance following acute ethanol administration of 1.0 g/kg ethanol (B) RR performance following acute ethanol administration of 2.0 g/kg ethanol, *p < 0.05, error bars denote S.E.M. (Post hoc test revealed no significant differences in effects between ages.)
AC C
EP
TE D
M AN U
SC
Figure 3. Mean blood ethanol concentration following 3.0 g/kg i.p. acute ethanol administration, *p < 0.05, error bars denote S.E.M.
ACCEPTED MANUSCRIPT
Mean RR Training (Last 3 Trials) (sec)
Mean RR Performance (sec)
Baseline
Saline
1.0 g/kg Ethanol
2.0 g/kg Ethanol
Adolescents
64.22 (15.46)
113.17 (31.32)
37.17 (15.29)
22.17 (7.06)
Adult
20.28 (4.72)
38.17 (14.35)
35.83 (14.40)
1 (0.63)
RI PT
Age Groups
AC C
EP
TE D
M AN U
SC
Aged 17.8 (17.00) 8 (1.88) 5.5 (2.91) 0 (0.00) Table 1. Mean performance times of animals on the accelerating rotarod. Baseline scores represent the average of the final 3 training trails. Data reported are means with standard error of the mean inside parentheses.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0741-8329(14)20208-4
DOI:
10.1016/j.alcohol.2014.12.002
Reference:
ALC 6480
To appear in:
Alcohol
Received Date: 10 October 2014 Revised Date:
1 December 2014
Accepted Date: 5 December 2014
Please cite this article as: Ornelas L.C., Novier A., Van Skike C.E., Diaz-Granados J.L. & Matthews D.B., The Effects of Acute Alcohol on Motor Impairments in Adolescent, Adult, and Aged Rats, Alcohol (2015), doi: 10.1016/j.alcohol.2014.12.002. 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.
ACCEPTED MANUSCRIPT 1
RI PT
The Effects of Acute Alcohol on Motor Impairments in Adolescent, Adult, and Aged Rats
Laura C. Ornelas1, Adelle Novier1, Candice E. Van Skike1, Jaime L. Diaz-Granados1, Douglas B. Matthews1,2
Baylor University, One Bear Place #97334, Waco, Texas 76798, USA
2
University of Wisconsin – Eau Claire, HHH273, Eau Claire, WI 54702, USA
AC C
EP
TE D
Please direct all correspondence to: Douglas B. Matthews, PhD University of Wisconsin – Eau Claire Department of Psychology Eau Claire, WI 54702 Telephone: +1 715 836 2119 Fax: +1 715 836 2124 Email: [email protected]
M AN U
SC
1
ACCEPTED MANUSCRIPT 2
Abstract Acute alcohol exposure has been shown to produce differential motor impairments between aged and adult rats and between adolescent and adult rats. However, the effects of acute
RI PT
alcohol exposure among adolescent, adult, and aged rats have yet to be systematically investigated within the same project using a dose-dependent analysis. We sought to determine the age- and dose-dependent effects of acute alcohol exposure on gross and coordinated motor
SC
performance across the rodent lifespan. Adolescent (PD 30), adult (PD 70), and aged
(approximately 18 months) male Sprague-Dawley rats were tested on 3 separate motor tasks:
M AN U
aerial righting reflex (ARR), accelerating rotarod (RR), and loss of righting reflex (LORR). In a separate group of animals, blood ethanol concentrations (BEC) were determined at multiple time points following a 3.0 g/kg ethanol injection. Behavioral tests were conducted with a Latin square repeated-measures design in which all animals received the following doses: 1.0 g/kg or
TE D
2.0 g/kg alcohol or saline over 3 separate sessions via intraperitoneal (i.p.) injection. During testing, motor impairments were assessed on the RR 10 min post-injection and on ARR 20 min post-injection. Aged animals spent significantly less time on the RR when administered 1.0 g/kg
EP
alcohol compared to adult rats. In addition, motor performance impairments significantly increased with age after 2.0 g/kg alcohol administration. On the ARR test, aged rats were more
AC C
sensitive to the effects of 1.0 g/kg and 2.0 g/kg alcohol compared to adolescents and adults. Seven days after the last testing session, animals were given 3.0 g/kg alcohol and LORR was examined. During LORR, aged animals slept longer compared to adult and adolescent rats. This effect cannot be explained solely by BEC levels in aged rats. The present study suggests that acute alcohol exposure produces greater motor impairments in older rats when compared to adolescent and adult rats and begins to establish a procedure to determine motor effects by alcohol across the lifespan.
ACCEPTED MANUSCRIPT 3
Highlights: Acute alcohol impairs motor performance more in aged rats than in younger rats.
•
Aged animals are more sensitive to the hypnotic effects of alcohol.
•
Aerial righting reflex is a more reliable measure of motor impairments than rotarod.
RI PT
•
AC C
EP
TE D
M AN U
SC
Keywords: alcohol, aerial righting reflex, rotarod, aging, loss of righting reflex
ACCEPTED MANUSCRIPT 4
The average age of the world’s population is rapidly rising (Lutz, Sanderson, & Scherbov, 2008). According to the United States Census Bureau, individuals aged 65 and older are projected to total approximately 72 million people and represent nearly 20% of the total U.S.
RI PT
population in the year 2030 (He, Sengupta, Velkoff, & DeBarros, 2005). Importantly,
approximately 50% of the elderly population (aged over 65) and almost 25% of individuals over 85 years old currently drink alcohol (Caputo et al., 2012). In addition, nearly 13% of men and
SC
8% of women over the age of 65 consume alcohol in a binge-drinking manner (Blazer & Wu, 2009). In the United States, it is estimated that the prevalence of alcohol-use disorders among the
M AN U
elderly population is approximately 1–3% (Caputo et al., 2012). As the global population continues to increase, substantial alcohol consumption and its consequences in the elderly are an important, but understudied, public health concern (Babor, 2010).
Individuals aged 65 years and older are especially susceptible to the risk factors
TE D
associated with alcohol consumption. Increased alcohol consumption in aged individuals could be a contributing factor to cognitive deficits including dementia (Thomas & Rockwood, 2001), and to impaired motor coordination and increased falls in older individuals (Høidrup, Grønbæk,
EP
Gottschau, Lauritzen, & Schroll, 1999; Jones, Cyr, & Patil, 1994; Mukamal et al., 2004; Mukamal, Robbins, Cauley, Kern, & Siscovick, 2007; Weafer & Fillmore, 2012). Deficits in
AC C
motor coordination in this age group may be related to age-dependent changes in cerebellar function (Piguet et al., 2006). Alcohol has been shown to decrease cell density and size of the cerebellar vermis, resulting in gait ataxia (Johnson-Greene et al., 1997; Phillips, Harper, & Kril, 1990; Piguet et al., 2006). Therefore, the aging cerebellum paired with heavy alcohol consumption could lead to increased motor impairments in the elderly.
ACCEPTED MANUSCRIPT 5
In concordance with age-related effects of alcohol in humans, research using animal models has shown that many of the effects of alcohol are age-dependent (Chin, Van Skike, & Matthews, 2010 for review). Adolescent rats, compared to adult rats, have been shown to be less
RI PT
sensitive to the sedative (Little, Kuhn, Wilson, & Swartzwelder, 1996), anxiogenic (Doremus, Brunell, Varlinskaya, & Spear, 2003), hypnotic (Matthews, Tinsley, Diaz-Granados, Tokunaga, & Silvers, 2008; Silveri & Spear, 1998), and motor impairing effects of acute alcohol (Ramirez
SC
& Spear, 2010; Van Skike et al., 2010; White et al., 2002). Furthermore, adolescent rodents exhibit significantly more acute tolerance compared to adult rodents (Draski, Bice, & Deitrich,
M AN U
2001; Grieve & Littleton, 1979; Silveri & Spear, 1998, 2002; Varlinskaya & Spear, 2006) and are more sensitive to alcohol-induced hypothermia (Ristuccia & Spear, 2008). Research indicates that older rodents are more sensitive to acute alcohol exposure compared to adolescent and adult rats (Novier, Van Skike, Diaz-Granados, Mittleman, &
TE D
Matthews, 2013; Van Skike et al., 2010). Older rodents are more sensitive to the hypnotic (Ott, Hunter, & Walker, 1985) and hypothermic effects of acute alcohol (Wood & Armbrecht, 1982), as well as the severity of alcohol withdrawal (Wood, Armbrecht, & Wise, 1982) compared to
EP
younger rodents. To explore the effects of alcohol across the lifespan, Van Skike et al. (2010) examined the impact of a single acute alcohol dose on motor impairments in 4 rodent age groups.
AC C
Aged rats showed significantly more impairment in motor performance compared to periadolescent and adolescent rats. Furthermore, Novier et al. (2013) investigated the differences in the motor and memory-impairing effects of acute alcohol between adult and aged rats. Similarly, results indicate that aged animals performed significantly worse in all behavioral measures compared to adult rats.
ACCEPTED MANUSCRIPT 6
Although current research has recognized differential motor impairments between adolescent and aged rats (Van Skike et al., 2010; White et al., 2002) and adult and aged rats (Novier et al., 2013), research has yet to systematically investigate the effect of acute ethanol on
RI PT
motor impairments across the lifespan using a dose-dependent analysis. In addition, the effect of age on a high dose of alcohol-induced hypnosis has yet to be investigated. Therefore, we sought to determine the age- and dose-dependent effects of acute alcohol exposure on gross and
SC
coordinated motor performance in adolescent, adult, and aged rats using the accelerated rotarod (RR) and aerial righting reflex (ARR). In addition, the effect of 3.0 g/kg on ethanol-induced loss
M AN U
of righting reflex (LORR) was determined. Finally, blood ethanol concentrations (BEC) were determined in a separate group of animals at 7 different time points following a 3.0 g/kg ethanol injection to better understand how BEC affects LORR at 3 different ages. We present evidence that alcohol produces greater motor impairments in older rats when compared to adolescent and
concentrations.
Subjects
EP
Materials and methods
TE D
adult rats and these motor impairments are not completely explained by blood ethanol
Twelve adolescent (postnatal day (PD) 28), 12 young adult (approximately PD 70), and
AC C
12 aged (approximately 18 months) male Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). These ages were selected because PD 28–30 is a developmental period of early adolescence based on evidence that mature sperm is not yet found, while all sperm are mature at PD 70, indicating this age is the beginning of adulthood (Odell, 1990). Aged animals were 18 months, to be consistent with our previous work (Novier et al., 2013). Animal care procedures were approved by the Institutional Animal Care and Use Committee of Baylor University. Animals were individually housed and given ad libitum access to food and water
ACCEPTED MANUSCRIPT 7
throughout the experiment. Following previously published methods, all rats acclimated to the colony room for 2 days before any experimental procedures (Chin et al., 2011; Novier, Van Skike, Chin, Diaz-Granados, & Matthews, 2012; Novier et al., 2013; Silvers et al., 2006;
RI PT
Tokunaga, Silvers, & Matthews, 2006; Van Skike, Novier, Diaz-Granados, & Matthews, 2012). To investigate the dose-dependent effects of acute ethanol exposure in the same animals and therefore reduce subject number, animals were involved in a Latin Square repeated-measures
SC
design with experimental doses of 1.0 g/kg or 2.0 g/kg (10% w/v) alcohol, or a saline dose
equivalent to the volume of a 1.0 g/kg dose of alcohol. Animals were first tested on the RR at
M AN U
10 min post ethanol or saline injection, and ARR was assessed 20 min post ethanol or saline injection. Three separate trials were conducted 3 days apart to minimize carryover effects. The first test session occurred 24 h after the last RR training trial (see below). Animals were randomly assigned to each of the 3 different drug orders and counterbalanced by age and dose
TE D
for all 3 trials. Seven days after the last testing trial, a subset of animals (n = 4 per age group) were given an acute injection of 3.0 g/kg alcohol and loss of righting (LORR) was determined. Finally, a separate group of adolescent, adult, and aged animals (n = 6 per age) were injected i.p.
EP
with 3.0 g/kg ethanol. The tail was nicked and blood was collected for BEC analysis via the Analox AM1 protocol 30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min post-
AC C
injection.
Aerial righting reflex (ARR)
The effect of acute alcohol exposure on gross motor impairment was assessed 20 min
after saline or alcohol administration by ARR as previously described (Novier et al., 2013; Van Skike et al., 2010). The 3 test trials occurred on PD 31, PD 35, and PD 39 in adolescent rats, and on PD 73, PD 77, and PD 81 in adult rats. Aged animals are classified as approximately
ACCEPTED MANUSCRIPT 8
18 months old by the supplier and therefore exact ages are unknown. A ruler was vertically taped above a 10-inch foam pad. Animals were initially released 5 inches (12.7 cm) above the foam pad in a supine position. An animal’s righting reflex was considered successful if 3 out of 4 paws
RI PT
made direct contact with the foam pad on 2 out of 3 releases. If righting was not successful, the height of release was increased in 5-inch (12.7 cm) increments up to a maximum height of 25 inches (63.5 cm). Subjects who failed to achieve successful righting reflex at 25 inches
SC
(63.5 cm) were given a score of 30 inches (76.2 cm) for statistical analysis. Accelerating rotarod (RR)
M AN U
Apparatus
RR was used to investigate the effects of alcohol exposure on motor coordination. Motor activity was tested on a 4-station Rota-Rod treadmill (Model ENV 575, Med Associates, St. Albans, VT). The apparatus was located in a behavioral room isolated from animal caging
Training
TE D
and housing.
Subjects received 5 consecutive training trials on the RR, as previously described (Novier
EP
et al., 2013). Adolescents received RR training on PD 30 and adults on PD 72. The rod accelerated from 4 rpm to 40 rpm over a 5-min period. The rotarod is covered with fine grit
AC C
sandpaper to provide a uniform surface and to reduce slipping (Rustay, Wahlsten, & Crabbe, 2003). The RR was interfaced to a computer that collected the time each subject remained on the rod up to a maximum time of 360 sec. After the 5 training trials were complete, averages of the last 3 trials were calculated and animals who failed to meet a criterion of 7 sec were given additional training trials until their final 3 training trials averaged 7 sec. Testing
ACCEPTED MANUSCRIPT 9
Subjects received an i.p. injection of 1.0 g/kg alcohol, 2.0 g/kg alcohol, or a saline equivalent dose. Motor activity on the RR was recorded 10 min after alcohol or saline administration. The 3 test trials occurred on PD 31, PD 35, and PD 39 in adolescent rats, and on
RI PT
PD 73, PD 77, and PD 81 in adult rats. Aged animals are classified as approximately 18 months old by the supplier and therefore exact ages are unknown. Loss of Righting Reflex (LORR)
SC
Procedure
The sedative/hypnotic effect of alcohol was assessed using the loss of righting reflex
M AN U
paradigm. One week after the last testing trial (PD 46 for adolescents and PD 88 for adults), a subset of previously tested animals (n = 4) received an i.p. injection of 3.0 g/kg alcohol. Animals were monitored throughout LORR. Latency to regain righting reflex was assessed. Recovery of reflex was defined as successfully righting 3 times in 1 min.
TE D
Blood ethanol concentration Procedure
To better understand how age impacts blood ethanol levels, 6 adolescent rats, 6 adult rats,
EP
and 6 aged rats were injected with 3.0 g/kg ethanol i.p. before collection of blood via the tail. Animals were the same age as those previously assessed for LORR to allow for a comparison of
AC C
how BEC influences righting reflex. Following injection, the tail was nicked and blood collected at 7 different time points (30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min) and BEC was assessed via the AM1 Analox system following manufacturer’s recommendations. Results Aerial Righting Reflex A two-way repeated measure ANOVA revealed a significant Age × Dose interaction, F(4,15) = 6.79, p < 0.05. To further investigate the significant interaction, one-way ANOVAs for
ACCEPTED MANUSCRIPT 10
age were performed at each of the 3 doses tested. Importantly, no significant differences by age were found when subjects were tested with saline, F(2,15) = 1.00, p = 0.39, indicating no baseline or carry over effects in performance (see Fig. 1A). A significant effect was found when
RI PT
animals were tested with 1.0 g/kg alcohol, F(2,15) = 9.80, p < 0.05. Bonferroni post hoc tests revealed that aged rats were more sensitive to the effects of 1.0 g/kg alcohol compared to adolescents, t = 3.84, p < 0.05, and adults, t = 3.84, p < 0.05 (see Fig. 1B). Similarly, a
SC
significant effect was found when rats were tested with 2.0 g/kg alcohol, F(2,15) = 10.19,
p < 0.05. Specifically, aged animals performed worse on ARR compared to adolescent, t = 4.37,
M AN U
p < 0.05, and adult rats, t = 3.17, p < 0.05 following the 2.0 g/kg dose (see Fig. 1C). Finally, adolescent and adult rats did not significantly differ in ARR performance when given 1.0 g/kg alcohol, t = 0.00, p > 0.05 or 2.0 g/kg alcohol, t = 1.20, p > 0.05. Accelerating rotarod
TE D
There was a significant main effect of age during RR training, F(2,15) = 7.57, p < 0.05. Bonferroni post hoc tests revealed a significant difference between adolescent and aged rats, t = 3.50, p < 0.05, and between adolescent and adult rats in training performance, t = 3.22,
EP
p < 0.05 (see Table 1). However, adult and aged animals did not significantly differ from one another on RR training performance, t = 0.29, p > 0.05. To equate for differential baseline
AC C
performance, the effect of acute alcohol on RR performance was analyzed by percent change from each animal’s baseline performance (see Table 1 for untransformed data). A two-way repeated measures ANOVA revealed a significant Age × Dose interaction
during RR testing, F(4,15) = 11.76, p < 0.05. One-way ANOVAs were performed to assess the significant differences between each individual dose tested. A significant effect was found when animals were tested with 1.0 g/kg alcohol, F(2,15) = 6.51, p < 0.05. Bonferroni post hoc tests
ACCEPTED MANUSCRIPT 11
showed aged animals performed worse on RR compared to adult rats when administered 1.0 g/kg alcohol, t = 3.46, p < 0.05 (see Fig. 2A). A significant effect was found when animals were tested with 2.0 g/kg alcohol, indicating motor performance impairments increased with age,
RI PT
F(2,15) = 4.28, p < 0.05 (however, post hoc test revealed no significant effects between ages) (see Fig. 2B). A one-way ANOVA revealed no significant differences between ages when tested
SC
with saline, F(2,15) = 2.80, p > 0.05. Loss of righting reflex
A one-way ANOVA revealed a significant difference in sleep time, F(2,9) = 8.02,
M AN U
p < 0.05. Bonferroni post hoc analysis showed aged animals slept longer (mean sleep time of 157.0 min and a standard error of the mean of 53.0 min) compared to adult (mean sleep time of 0 min and a standard error of the mean of 0 min), t = 3.56, p < 0.05, and adolescent rats (mean sleep time of 8.25 min and a standard error of the mean of 8 min), t = 3.37, p < 0.05. No
p > 0.05.
TE D
significant difference in sleep time was found between adolescent and adult rats, t = 0.19,
Blood ethanol concentration
EP
A two-way ANOVA revealed a significant Age × Time interaction on blood ethanol
AC C
concentration, F(12,90) = 23.41, p < 0.001. One-way ANOVAs were performed to assess the significant differences at each time tested. Adolescent blood ethanol levels were significantly greater 30 min post-injection compared to both adult and aged animals (Tukey post hoc comparisons, both p's < 0.05). However, adolescent blood ethanol concentrations were significantly less than adults (p < 0.05) and aged animals (p < 0.001) at the 240-, 300-, and 360-min time points. Finally, adult blood ethanol concentrations were significantly different from aged animals 360 min post-ethanol injection (p < 0.001) (see Fig. 3).
ACCEPTED MANUSCRIPT 12
Discussion The current study examined the age-and dose-dependent effects of acute alcohol exposure on gross and coordinated motor performance across the rodent lifespan and provides a
RI PT
partial ethanol clearance curve following a high-dose ethanol administration. On the ARR and RR tasks, aged rats exhibited greater deficits in motor performance compared to both adolescent and adult rats during acute alcohol exposure at both doses. In addition, aged animals slept longer
SC
compared to both adult and adolescent rats in the LORR task. To our knowledge, this study is the first to report a difference in motor impairments and sedative/hypnotic effects of acute alcohol
following a high-dose ethanol exposure.
M AN U
among adolescent, adult, and aged rats and provides novel data concerning blood ethanol levels
Research from our laboratory has investigated the age-dependent effects of acute alcohol exposure on motor impairments (Novier et al., 2013; Van Skike et al., 2010). The results of the
TE D
present study are in accordance with previous research that shows age-dependent impairments in ARR performance after acute alcohol administration (Van Skike et al., 2010). However, Van Skike et al. (2010) reported adult and aged rats did not significantly differ from each other
EP
during the ARR paradigm, whereas in the present set of studies aged animals were significantly more impaired relative to adults. A possible explanation for this discrepancy is our adult rats
AC C
were tested between PND 73 and PND 88, while Van Skike et al. (2010) tested adult rats at PD 120. In addition, Van Skike et al. (2010) assessed gross motor performance using only the ARR task. The current study included the RR as an additional measure of motor skill learning to investigate the age-dependent differences in motor performance associated with acute alcohol exposure prior to the ARR task. It is possible that the sequential nature of the testing procedure in the current set of studies altered ARR performance and may have produced greater ARR
ACCEPTED MANUSCRIPT 13
deficits in aged subjects in the current experiments. Future research using multiple measures of behavior within a single ethanol dose needs to address potential sequential effects. Novier et al. (2013) found that aged animals showed greater alcohol-induced ataxia on
RI PT
the RR compared to adult rats when animals were tested with moderate to high alcohol doses. However, Novier et al. (2012) found that adolescent and adult animals had facilitated RR performance following administration of a low (1.0 g/kg) dose of alcohol, a result that is
SC
replicated for adult animals but not adolescent animals in the current work. Furthermore, the current work is the first to investigate both low (1.0 g/kg) and moderately high (2.0 g/kg) doses
M AN U
of ethanol on the rotarod across a full age range. The current work extends previous findings to demonstrate that aged animals are still impaired on the rotarod at a low dose of alcohol and this impairment is magnified as the dose of ethanol increases. Additionally, in agreement with results from the present study, Novier et al. (2013) reported no significant differences between adult and
TE D
aged rats in RR training performance. However, the experimental procedures for RR training were slightly different compared to our previous studies (Novier et al., 2013). Due to a small sample size, animals who failed to meet criterion at 7 sec in the present study were given
EP
additional training trials until their final 3 training trials averaged 7 sec, rather than being excluded from RR testing as was done previously. Such a training procedure may bias our results
AC C
and confound a potential impairment due to a floor effect. Future studies should use a slower acceleration speed or a fixed speed task to determine if aged animals are indeed more impaired by acute ethanol on this task. Finally, aged animals slept longer compared to adolescent and adult rats during LORR.
These results are consistent with previous studies, which state that adolescent animals are less sensitive to alcohol-induced sedative/hypnotic effects (Little et al., 1996; Matthews et al., 2008;
ACCEPTED MANUSCRIPT 14
Silveri & Spear, 1999). While BECs were not taken from animals when they regained their righting reflex, we did investigate BEC in a separate group of same-aged adolescent, adult, and aged animals following a 3.0 g/kg ethanol injection. Initially, adolescent animals demonstrate
RI PT
higher BEC than either adult or aged animals, but adolescents have lower BEC compared to the other 2 age groups following the 240-min time point until the last blood collection time. Such a time-dependent interaction of age and BEC is intriguing and suggests that behaviors impacted by
SC
alcohol may also show a time-dependent effect when different ages are tested at different times in the BEC curve. Furthermore, the BEC data provide some important insight into the LORR
M AN U
data as it relates to the question of whether aged animals are more sensitive to a high dose of alcohol than adolescent or adult animals. Specifically, aged rats slept approximately 150 min following administration of 3.0 g/kg ethanol, a time point when the BEC clearance study suggests blood ethanol levels among the 3 ages should be similar (no significant difference in
TE D
BEC at either the 120-min or 180-min time point between adolescent, adult, and aged rats). However, without determining BEC in subjects undergoing the LORR experiment, future research will need to determine if aged animals are indeed more sensitive to acute ethanol on the
EP
LORR test. Furthermore, BEC in aged animals remained high throughout the experiment even though collection times occurred 6 h after injection. This suggests that the aged system is
AC C
impaired in clearance of the drug and future studies should address this issue across a full ethanol dose range. In addition, there is a lack of ethanol clearance in the adult animals until the final time point. Such a lack of clearance was unpredicted and highlights the potential limitation of using i.p. injections to study the effects of ethanol and the need to collect BEC levels from animals used in future LORR studies. Finally, the impact of sex and age on ethanol’s effects is
ACCEPTED MANUSCRIPT 15
understudied, particularly for aged animals. Future studies should include female subjects to better understand how sex and age interact. The continuous decline of motor function is a widespread feature of aging (Desrosiers,
RI PT
Hébert, Bravo, & Dutil, 1995; Ranganathan, Siemionow, Sahgal, & Yue, 2001; Smith et al., 1999). Age-related deficits in motor function may be the consequence of age-related muscle deterioration, or sarcopenia (Herndon et al., 2002), leading to impaired mobility, gait
SC
abnormalities, and increased risk of falls (Hunter, White, & Thompson, 1998). Previous research has indicated that aged rats have deficits in motor coordination, balance, and reflexes as
M AN U
indicated by the RR and other tasks of motor performance (Shukitt-Hale, Mouzakis, & Joseph, 1998; Wallace, Krauter, & Campbell, 1980). These deficits may be associated with agedependent decline in strength and muscle mass (Hepple, Ross, & Rempfer, 2004). In addition, alcohol metabolism changes with age, which may result in aged humans being more sensitive to
TE D
the toxic effects of alcohol (Seitz et al., 1993). Seitz et al. (1993) reported gastric alcohol dehydrogenase (ADH) activity was significantly lower in elderly male subjects. Decreased ADH may contribute to reduced first pass metabolism, leading to an increase in blood alcohol
EP
concentrations as well as impairments in motor coordination (Seitz et al., 1993). This raises the possibility that aged animals in the current project are more impaired by acute alcohol due to
AC C
differential BECs (see Fig. 3). Although we did not measure BEC following the lower injection amounts of 1.0 g/kg and 2.0 g/kg ethanol, we do not think differential BEC levels explain the current data for two reasons. First, alcohol was administered i.p. and therefore different ADH activity in the gut would not alter BEC levels. Second, previous work from our laboratory has shown, using identical ages and the same lower alcohol doses, that while alcohol produces greater cognitive and motor impairment in aged animals compared to adult animals, BEC did not
ACCEPTED MANUSCRIPT 16
predict greater alcohol impairment in aged animals (Novier et al., 2013). However, when feasible, future projects should continue to monitor BEC in studies investigating the effect of alcohol across different ages.
RI PT
The present study provides evidence that the ARR paradigm appears to be a better
assessment of motor performance compared to the RR task. Animals’ performance on the ARR task was consistent throughout each testing trial and provided less variability in motor
SC
performance compared to RR. In addition, there is no significant baseline difference. A possible explanation as to why the RR produced differences in motor impairments compared to the ARR
M AN U
could be the specific design of the RR. The size of the cylindrical rotating rod was the same size in diameter for all 3 age groups tested. In addition, the acceleration of the rod might not be the best procedure to test performance in this task. Future studies that use the RR to assess motor performance on aged animals should adjust RR parameters to accommodate changes in body
TE D
weight and size of the animals.
In conclusion, the results of the present study show that acute alcohol administration produces age- and dose-dependent effects on motor performance. With the inclusion of 3
EP
different developmental age groups, the current study provides a more comprehensive view of age-dependent motor impairments during the rodent lifespan. The present study adds to the
AC C
growing body of research that indicates alcohol produces age-dependent impairments in motor performance.
ACCEPTED MANUSCRIPT 17
References
RI PT
Babor, T. (ed.). (2010). Alcohol: No Ordinary Commodity: Research and Public Policy. (Oxford: Oxford University Press).Blazer, D. G., & Wu, L. T. (2009). The epidemiology of at-risk and binge drinking among middle-aged and elderly community adults: National Survey on Drug Use and Health. The American Journal of Psychiatry, 166, 1162–1169.Caputo, F., Vignoli, T., Leggio, L., Addolorato, G., Zoli, G., & Bernardi, M. (2012). Alcohol use disorders in the elderly: a brief overview from epidemiology to treatment options. Experimental Gerontology, 47, 411–416. Chin, V. S., Van Skike, C. E., Berry, R. B., Kirk, R. E., Diaz-Granados, J., & Matthews, D. B. (2011). Effect of acute ethanol and acute allopregnanolone on spatial memory in adolescent and adult rats. Alcohol, 45, 473–483.
SC
Chin, V. S., Van Skike, C. E., & Matthews, D. B. (2010). Effects of ethanol on hippocampal function during adolescence: a look at the past and thoughts on the future. Alcohol, 44, 3– 14.
M AN U
Desrosiers, J., Hébert, R., Bravo, G., & Dutil, É. (1995). Upper extremity performance test for the elderly (TEMPA): normative data and correlates with sensorimotor parameters. Archives of Physical Medicine and Rehabilitation, 76, 1125–1129. Doremus, T. L., Brunell, S. C., Varlinskaya, E. I., & Spear, L. P. (2003). Anxiogenic effects during withdrawal from acute ethanol in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 75, 411–418.
TE D
Draski, L. J., Bice, P. J., & Deitrich, R. A. (2001). Developmental alterations of ethanol sensitivity in selectively bred high and low alcohol sensitive rats. Pharmacology, Biochemistry, and Behavior, 70, 387–396. Grieve, S. J., & Littleton, J. M. (1979). Age and strain differences in the rate of development of functional tolerance to ethanol by mice. The Journal of Pharmacy and Pharmacology, 31, 696–700.
EP
He, W., Sengupta, M., Velkoff, V. A., & DeBarros, K. A. (2005). 65+ in the United States, 2005. (Washington, DC: U.S. Government Printing Office).
AC C
Hepple, R. T., Ross, K. D., & Rempfer, A. B. (2004). Fiber atrophy and hypertrophy in skeletal muscles of late middle-aged Fischer 344 × Brown Norway F1-hybrid rats. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 59, 108–117. Herndon, L. A., Schmeissner, P. J., Dudaronek, J. M., Brown, P. A., Listner, K. M., Sakano, Y., et al. (2002). Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature, 419, 808–814. Høidrup, S., Grønbæk, M., Gottschau, A., Lauritzen, J. B., & Schroll, M. (1999). Alcohol intake, beverage preference, and risk of hip fracture in men and women. Copenhagen Centre for Prospective Population Studies. American Journal of Epidemiology, 149, 993–1001. Hunter, S., White, M., & Thompson, M. (1998). Techniques to evaluate elderly human muscle function: a physiological basis. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 53, B204–B216.
ACCEPTED MANUSCRIPT 18
Johnson-Greene, D., Adams, K. M., Gilman, S., Kluin, K. J., Junck, L., Martorello, S., et al. (1997). Impaired upper limb coordination in alcoholic cerebellar degeneration. Archives of Neurology, 54, 436–439. Jones, T. V., Cyr, D. G., & Patil, K. (1994). Effects of alcohol measured by dynamic posturography in elderly women. Journal of the American Geriatrics Society, 42, Sa72.
RI PT
Little, P. J., Kuhn, C. M., Wilson, W. A., & Swartzwelder, H. S. (1996). Differential effects of ethanol in adolescent and adult rats. Alcoholism: Clinical and Experimental Research, 20, 1346–1351. Lutz, W., Sanderson, W., & Scherbov, S. (2008). The coming acceleration of global population ageing. Nature, 451, 716–719.
SC
Matthews, D. B., Tinsley, K. L., Diaz-Granados, J. L., Tokunaga, S., & Silvers, J. M. (2008). Chronic intermittent exposure to ethanol during adolescence produces tolerance to the hypnotic effects of ethanol in male rats: a dose-dependent analysis. Alcohol, 42, 617–621.
M AN U
Mukamal, K. J., Mittleman, M. A., Longstreth, W. T., Newman, A. B., Fried, L. P., & Siscovick, D. S. (2004). Self‐reported alcohol consumption and falls in older adults: cross‐sectional and longitudinal analyses of the cardiovascular health study. Journal of the American Geriatrics Society, 52, 1174–1179. Mukamal, K. J., Robbins, J. A., Cauley, J. A., Kern, L. M., & Siscovick, D. S. (2007). Alcohol consumption, bone density, and hip fracture among older adults: the cardiovascular health study. Osteoporosis International, 18, 593–602.
TE D
Novier, A., Van Skike, C. E., Chin, V. S., Diaz-Granados, J. L., & Matthews, D. B. (2012). Low and moderate doses of acute ethanol do not impair spatial cognition but facilitate accelerating rotarod performance in adolescent and adult rats. Neuroscience Letters, 512, 38–42.
EP
Novier, A., Van Skike, C. E., Diaz‐Granados, J. L., Mittleman, G., & Matthews, D. B. (2013). Acute alcohol produces ataxia and cognitive impairments in aged animals: a comparison between young adult and aged rats. Alcoholism: Clinical and Experimental Research, 37, 1317–1324.
AC C
Odell, W. D. (1990). Sexual maturation in the rat. In Control of the Onset of Puberty, M. M. Grumback, P. C. Sizonenko, & M. L. Aubert, eds. (Baltimore, MD: Williams and Williams), pp. 183–210. Ott, J. F., Hunter, B. E., & Walker, D. W. (1985). The effect of age on ethanol metabolism and on the hypothermic and hypnotic responses to ethanol in the Fischer 344 rat. Alcoholism: Clinical and Experimental Research, 9, 59–65. Phillips, S. C., Harper, C. G., & Kril, J. J. (1990). The contribution of Wernicke's encephalopathy to alcohol-related cerebellar damage. Drug and Alcohol Review, 9, 53– 60. Piguet, O., Cramsie, J., Bennett, H. P., Kril, J. J., Lye, T. C., Corbett, A. J., et al. (2006). Contributions of age and alcohol consumption to cerebellar integrity, gait and cognition in non-demented very old individuals. European Archives of Psychiatry and Clinical Neuroscience, 256, 504–511.
ACCEPTED MANUSCRIPT 19
Ramirez, R. L., & Spear, L. P. (2010). Ontogeny of ethanol-induced motor impairment following acute ethanol: assessment via the negative geotaxis reflex in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 95, 242–248. Ranganathan, V. K., Siemionow, V., Sahgal, V., & Yue, G. H. (2001). Effects of aging on hand function. Journal of the American Geriatrics Society, 49), 1478–1484.
RI PT
Ristuccia, R. C., & Spear, L. P. (2008). Autonomic responses to ethanol in adolescent and adult rats: a dose–response analysis. Alcohol, 42, 623–629. Rustay, N. R., Wahlsten, D., & Crabbe, J. C. (2003). Influence of task parameters on rotarod performance and sensitivity to ethanol in mice. Behavioural Brain Research, 141, 237– 249.
SC
Seitz, H. K., Egerer, G., Simanowski, U. A., Waldherr, R., Eckey, R., Agarwal, D. P., et al. (1993). Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism. Gut, 34, 1433–1437.
M AN U
Shukitt-Hale, B., Mouzakis, G., & Joseph, J. A. (1998). Psychomotor and spatial memory performance in aging male Fischer 344 rats. Experimental Gerontology, 33, 615–624. Silveri, M. M., & Spear, L. P. (1998). Decreased sensitivity to the hypnotic effects of ethanol early in ontogeny. Alcoholism: Clinical and Experimental Research, 22, 670–676. Silveri, M. M., & Spear, L. P. (1999). Ontogeny of rapid tolerance to the hypnotic effects of ethanol. Alcoholism: Clinical and Experimental Research, 23, 1180–1184.
TE D
Silveri, M. M., & Spear, L. P. (2002). The effects of NMDA and GABAA pharmacological manipulations on ethanol sensitivity in immature and mature animals. Alcoholism: Clinical and Experimental Research, 26, 449–456. Silvers, J. M., Tokunaga, S., Mittleman, G., O'Buckley, T., Morrow, A. L. & Matthews, D. B. (2006). Chronic intermittent ethanol exposure during adolescence reduces the effect of ethanol challenge on hippocampal allopregnanolone levels and Morris water maze task performance. Alcohol, 39, 151–158.
EP
Smith, C. D., Umberger, G. H., Manning, E. L., Slevin, J. T., Wekstein, D. R., Schmitt, F. A., et al. (1999). Critical decline in fine motor hand movements in human aging. Neurology, 53, 1458–1461.
AC C
Thomas, V. S., & Rockwood, K. J. (2001). Alcohol abuse, cognitive impairment, and mortality among older people. Journal of the American Geriatrics Society, 49, 415–420. Tokunaga, S., Silvers, J. M., & Matthews, D. B. (2006). Chronic intermittent ethanol exposure during adolescence blocks ethanol-induced inhibition of spontaneously active hippocampal pyramidal neurons. Alcoholism: Clinical and Experimental Research, 30, 1–6. Van Skike, C. E., Botta, P., Chin, V. S., Tokunaga, S., McDaniel, J. M., Venard, J., et al. (2010). Behavioral effects of ethanol in cerebellum are age dependent: potential system and molecular mechanisms. Alcoholism: Clinical and Experimental Research, 34, 2070– 2080.
ACCEPTED MANUSCRIPT 20
Van Skike, C. E., Novier, A. K., Diaz-Granados, J. L., & Matthews, D. B. (2012). The effect of chronic intermittent ethanol exposure on spatial memory in adolescent rats: The dissociation of metabolic and cognitive tolerance. Brain Research, 1453, 34–39.
RI PT
Varlinskaya, E. I., & Spear, L. P. (2006). Ontogeny of acute tolerance to ethanol‐induced social inhibition in Sprague–Dawley rats. Alcoholism: Clinical and Experimental Research, 30, 1833–1844. Wallace, J. E., Krauter, E. E., & Campbell, B. A. (1980). Motor and reflexive behavior in the aging rat. Journal of Gerontology, 35, 364–370.
SC
Weafer, J., & Fillmore, M. T. (2012). Acute tolerance to alcohol impairment of behavioral and cognitive mechanisms related to driving: drinking and driving on the descending limb. Psychopharmacology (Berl), 220, 697–706. White, A. M., Truesdale, M. C., Bae, J. G., Ahmad, S., Wilson, W. A., Best, P. J., et al. (2002). Differential effects of ethanol on motor coordination in adolescent and adult rats. Pharmacology, Biochemistry, and Behavior, 73, 673–677.
M AN U
Wood, W. G., & Armbrecht, H. J. (1982). Behavioral effects of ethanol in animals: age differences and age changes. Alcoholism: Clinical and Experimental Research, 6, 3–12.
AC C
EP
TE D
Wood, W. G., Armbrecht, H. J., & Wise, R. W. (1982). Ethanol intoxication and withdrawal among three age groups of C57BL/6NNIA mice. Pharmacology, Biochemistry, and Behavior, 17, 1037–1041.
ACCEPTED MANUSCRIPT 21
Figure captions Figure 1. Aged animals are more sensitive to the gross motor effects of acute ethanol compared to adolescent and adult rats. (A) ARR height achieved after saline (B) ARR height achieved after 1.0 g/kg ethanol (C) ARR height achieved after 2.0 g/kg ethanol, *p < 0.05, error bars denote S.E.M.
RI PT
Figure 2. Coordinated motor impairments on RR increased with age following acute ethanol administration. (A) RR performance following acute ethanol administration of 1.0 g/kg ethanol (B) RR performance following acute ethanol administration of 2.0 g/kg ethanol, *p < 0.05, error bars denote S.E.M. (Post hoc test revealed no significant differences in effects between ages.)
AC C
EP
TE D
M AN U
SC
Figure 3. Mean blood ethanol concentration following 3.0 g/kg i.p. acute ethanol administration, *p < 0.05, error bars denote S.E.M.
ACCEPTED MANUSCRIPT
Mean RR Training (Last 3 Trials) (sec)
Mean RR Performance (sec)
Baseline
Saline
1.0 g/kg Ethanol
2.0 g/kg Ethanol
Adolescents
64.22 (15.46)
113.17 (31.32)
37.17 (15.29)
22.17 (7.06)
Adult
20.28 (4.72)
38.17 (14.35)
35.83 (14.40)
1 (0.63)
RI PT
Age Groups
AC C
EP
TE D
M AN U
SC
Aged 17.8 (17.00) 8 (1.88) 5.5 (2.91) 0 (0.00) Table 1. Mean performance times of animals on the accelerating rotarod. Baseline scores represent the average of the final 3 training trails. Data reported are means with standard error of the mean inside parentheses.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
