Moderate chronic fetal alcohol exposure causes a motor learning deficit in adult outbred Swiss-Webster mice

Introduction

Ethanol (EtOH) is a toxin that produces variable detrimental physical and neurobehavioral effects in individuals subjected to ethanol exposure in utero, possibly resulting in Fetal Alcohol Spectrum Disorder (FASD). Animal models can replicate the human condition of FASD relatively well (Wilson & Cudd, 2011), but the broad spectrum of human drinking choices made during pregnancy is reflected in the enormous number of different FASD models currently employed by researchers. Even within a single species, differences in acute versus chronic exposure, the timing of the exposure, route of administration, and forced versus voluntary drinking patterns can result in a vast array of possible phenotypes.

The use of inbred mice has decreased variability in a field with a vast variety of models and made it easier to compare data across paradigms, explore voluntary drinking paradigms, and identify specific genetic differences regulating individual sensitivity to ethanol (Mayfield et al., 2008). Genetic variation causes some strains of mice to voluntarily drink more than other strains (Rhodes et al., 2007) — For example, C57BL/6 mice are heavy drinkers, while DBA2J mice are abstainers (Rhodes et al., 2005). Many FASD studies currently use inbred C57B/6 mice because of the reliability of consumption in this strain (Rhodes et al., 2005).

Inbred mouse strains exposed to chronic gestational ethanol have demonstrated deficits in motor function and learning/memory (Brady et al., 2012; Sanchez Vega et al., 2013). Typically, motor reflexes/coordination and learning/memory skills appear to be less dependent on the timing of alcohol exposure than activity and anxiety-related behavioral changes, which have more narrow windows (Mantha et al., 2013).

However, use of inbred strains do not faithfully replicate the genetic variation that naturally occurs within the human condition, so outbred strains also provide valuable information about FASD. Outbred Wistar rats are commonly used as a FASD rodent model, but use of outbred mouse models is much less common. Previously reported inbred alcohol-induced deficits may not show up in an outbred mouse model—for example, corticothalamic differences were not observed in an outbred mouse FASD model (White et al., 2015). But a recent paper characterized the effect of moderate prenatal ethanol exposure on outbred Swiss Webster mouse neonate behavior (including surface righting, negative geotaxis, cliff aversion, auditory startle, ear twitch, open field traversal, air righting, and eye opening) and found that ethanol-exposed pups achieved some developmental milestones (surface righting, cliff aversion, and open field traversal) at a different rate than non-exposed control pups (Chi et al., 2016).

To examine whether observed deficits in outbred mouse neonates can persist into adulthood, we examined the effect of chronic moderate gestational ethanol exposure on adult neurobehavioral outcomes in the offspring previously studied by Chi & colleagues (2016). We measured both learning/memory and motor skill subsets in male mice in order to better understand the behavioral impact of developmental ethanol exposure in an outbred model.

Materials and methods

Results

Open field

In open field testing, the amount of time spent in the open center square was not significantly different between ethanol-exposed and control animals (by t-test, p=0.2383; mean EtOH=13.72, SE=8.535; mean H₂O=6.973, SE=2.476; n=4 litters; Figure 1A). There was no significant difference in locomotor activity between ethanol-exposed and control mice, as measured by the mean number of squares traversed (by t-test, p=0.4231; mean EtOH=34.56, SE=9.691; mean H₂O=37.83, SE=7.781; n=4; Figure 1B).

Figure 1. Adult open field performance in Swiss Webster outbred mice exposed to moderate gestational ethanol.

(A) There was no significant difference found between ethanol-exposed mice and control mice in the average number of seconds spent in the center square of the open field over a 2 minute period, as a measure of anxiety-like behavior (by t-test, p=0.2383; mean EtOH=13.72, SE=8.535; mean H₂O=6.973, SE=2.476; n=4 litters). (B) No differences were found in the total number of squares crossed in the open field measure, as a measure of locomotion (by t-test, p=0.4231; mean EtOH=34.56, SE=9.691; mean H₂O=37.83, SE=7.781).

Barnes maze

Mice that were exposed to EtOH during development showed no differences on the probe day in total hole exploration (by t-test, p=0.7777; mean EtOH=6.96, SE=1.028; mean H₂O=7.67, SE=2.12; n=4 litters), number of incorrect hole explorations before finding the target hole (by t-test, p=0.6009; mean EtOH=5.79, SE=0.3560; mean H₂O=6.83, SE=1.766; n = 4), or the number of hole explorations after finding the target (by t-test, p=0.7352; mean EtOH=1.17, SE=0.7876; mean H₂O=0.83, SE=0.50; n=4; Figure 2A). There was also no significant difference found in the percentage of holes explored on the opposite quadrant of the maze (by t-test, p=0.1272; mean EtOH=33.33, SE=9.129; mean H₂O=57.50, SE=10.13; n= 4; Figure 2B).

Figure 2. Adult Barnes maze performance in Swiss Webster outbred mice exposed to moderate gestational ethanol.

(A) There was no significant difference between ethanol-exposed mice and control mice in number of mistakes made before finding the target hole (by t-test, p=0.6009; mean EtOH=5.79, SE=0.3560; mean H₂O=6.83, SE=1.766; n = 4 litters), the number of hole explorations after finding the target (by t-test, p=0.7352; mean EtOH=1.17, SE=0.7876; mean H₂O=0.83, SE=0.50), or the total hole explorations on the final probe day (by t-test, p=0.7777; mean EtOH=6.96, SE=1.028; mean H₂O=7.67, SE=2.12). (B) When the maze was divided into 4 quadrants for analysis, there was also no difference between the ethanol and control mice in the percent of holes located the opposite quadrant that were explored (by t-test, p=0.1272; mean EtOH=33.33, SE=9.129; mean H₂O=57.50, SE=10.13).

Kondziela Inverted Screen Test

There was no significant difference in grip strength between ethanol-exposed and control mice, as measured by the average latency to fall over the three trials (by t-test, p=0.855; mean EtOH=207.396 sec, SE=48.585; mean H₂O=193.39, SE=41.221; n=4 litters; Figure 3).

Figure 3. Adult inverted screen test performance in Swiss Webster outbred mice exposed to moderate gestational ethanol.

There was no significant difference found between ethanol-exposed mice and control mice in the latency to fall from the inverted screen as a measure of grip strength (by t-test, p=0.855; mean EtOH=207.396 seconds, SE=48.585; mean H₂O=193.39, SE=41.221; n=4 litters).

Rotarod

By the final performance day, previous ethanol exposure had no effect on the max RPM that mice were able to achieve (by t-test, p=0.399; mean EtOH=7.19, SE=0.68; mean H₂O=9.0, SE=1.78; n=4 litters; Figure 4A), indicating that overall motor performance after training was not affected by the gestational ethanol exposure. However, ethanol-exposed animals showed significantly less improvement between the first day of training and the final rotarod performance day, indicating a deficit in the acquisition of this motor skill (by t-test, p=0.008; mean EtOH=30.11% improvement, SE = 2.5%; mean H₂O=53.52%, SE=5.5%; Figure 4B).

Figure 4. Adult rotarod performance in Swiss Webster outbred mice exposed to moderate gestational ethanol.

(A) There was no significant difference between ethanol-exposed mice and control mice in the average max RPM attained on the final performance day (5) (by t-test, p=0.399; mean EtOH=7.19, SE=0.68; mean H₂O=9.0, SE=1.78; n=4 litters). (B) On average, control mice improved more than ethanol-exposed mice over the course of the rotarod testing, as measured by the percent change in RPM reached from the first training day (1) to the last performance day (5) (by t-test, p=0.008, EtOH mean=30.11%, SE=2.5%; H₂O mean=53.52%, SE=5.5%). (C) We found a possible effect of gestational ethanol exposure on the max RPM at which the mice were able to run over the course of the three training days (F_(2,12)=4.812, p=0.029), but this result was not significant after Sidak correction.

Both saline and ethanol treated mice showed an increase in the latency to fall in succeeding trial days at each age examined, indicating that both groups of mice were learning how to stay on the rod longer. Analysis by mixed ANOVA showed a significant effect of trial day over the three day training period [F_(1,6)=37.694; p=0.01; Figure 4C]. We found a possible effect of gestational ethanol exposure on the max RPM at which the mice were able to run over the course of the three training days (F_(2,12)=4.812, p=0.029; Figure 4C), but this result was not significant after Sidak correction.

Dataset 1.Raw data of motor learning deficit in adult FASD mice.

All raw data supporting the findings described in the paper are provided (Data files 1–5). The readme file contains descriptions of each data file.

Discussion

Our experiment showed that chronic low/moderate gestational ethanol exposure had no effect on some motor measures in adult outbred mice, including grip strength and overall rotarod performance, yet mice exposed to prenatal ethanol did not improve as quickly when learning the rotarod activity. This adult rotarod learning deficits may be due to long-term ethanol induced changes in specific brain areas that are involved in the acquisition of complex motor skills.

A recent meta-analysis of human FASD concluded that prenatal ethanol exposure can result in gross motor deficits, including gait and balance problems (Lucas et al., 2014). Rodent studies have indicated that ethanol exposure, particularly in the third trimester equivalent, can impact cerebellar Purkinje cell development and alter motor behavior (Maier et al., 1999). The cerebellum is traditionally held responsible for the coordination and execution of motor skills, particularly gait and balance. Ethanol-induced cerebellar damage can cause FASD motor skill deficits, and compromised corticocerebellar circuits may result in impaired visuospatial abilities or other more cognitive processes. However, in rodent models, cerebellar Purkinje cells seem particularly vulnerable to degeneration when exposed to alcohol during the critical period that occurs during the early postnatal period (human third trimester equivalent) (Jaatinen & Rintala, 2008), whereas our mouse model was only exposed during the first and second trimester equivalents.

The striatum (caudate/putamen) also plays a central role in the acquisition of long term motor skills, and remains of particular interest to alcohol researchers because of its central role in addictive behavior. In rodents, moderate gestational ethanol exposure can alter medium spiny neuron dendritic branching in the striatum pups (Rice et al., 2012), and studies show that developmental ethanol exposure can alter brain-derived neurotrophic factor (BDNF) expression in adult offspring, which can, in turn, impact propensity to drink and lay the groundwork for future addictive behavior, regulated by the striatum (Davis, 2008). BDNF is enriched in the striatum, delivered predominately via corticostriatal afferents, and BDNF polymorphisms have been linked to decreased synaptic plasticity involved in learning both complex and simple learning motor tasks (Cárdenas-Morales et al., 2014). It is possible that BDNF alterations in the striatum could impact synaptic plasticity or neurogenesis and could cause our observed complex motor learning changes.

Our experiment showed that chronic low/moderate gestational ethanol exposure had no effect on open field behavior or Barnes maze performance in outbred adult mice. Our results are in line with some previous studies that have not detected deficits that persist into adulthood for open field, learning/memory tasks, nor locomotor activity in inbred mouse models after chronic gestational ethanol exposure (Boehm et al., 2008; Downing et al., 2009), but it is also possible that our outbred strain introduces enough genetic variability to mask learning or open field phenotypes that have been previously found in inbred strains.

However, these ethanol-exposed offspring previously exhibited significantly altered timing to achieve surface righting, cliff aversion, and open field traversal developmental milestones as neonates (Chi et al., 2016), so our results may also indicate some sort of recovery or compensatory response by the central nervous system to the effects of early ethanol exposure has occurred. In most rodent FASD models, motor deficits are visible early in life and have disappeared by adulthood (Patten et al., 2014).

The value of non-inbred FASD models is currently being explored in other ways: High Alcohol Preferring mice and Low Alcohol Preferring mice have been developed by interbreeding eight inbred strains that display these drinking tendencies (Bice et al., 2011). Testing has shown differences in open field behavior between High and Low Alcohol Preferring mice in the absence of alcohol exposure (Can et al., 2012). Further behavioral characterization of these animals using an FASD model would provide useful motor behavior information, while decreasing the genetic variance present.

Future ethanol research should pay particular attention to adult phenotypes in order to document the persistence of these changes in FASD models, and to investigate compensatory mechanisms that may allow ethanol-exposed juveniles to overcome these deficits by adulthood. Neuroanatomical analysis should be performed at both juvenile and adult time points to detect possible apoptosis in brain areas involved in motor function. Apart from cell death, both cerebellar and striatal mechanisms that govern the execution of complex motor tasks should be examined, as well as possible long-lasting changes in synaptic plasticity or neurogenesis that may occur following developmental ethanol exposure.

Data availability

F1000Research: Dataset 1. Raw data of motor learning deficit in adult FASD mice, 10.5256/f1000research.9237.d130218 (Reekes et al., 2016).