Elsevier

NeuroToxicology

Volume 79, July 2020, Pages 25-39
NeuroToxicology

Full Length Article
Effects of prenatal ethanol exposure on acoustic characteristics of play fighting-induced ultrasonic vocalizations in juvenile rats

https://doi.org/10.1016/j.neuro.2020.03.016Get rights and content

Highlights

  • Prenatal high-ethanol exposure enhances the negative emotionality in juvenile rats.

  • Play fighting-induced 20–35 kHz negative ultrasonic vocalizations (USVs) are elevated in high-ethanol males.

  • Reductions in positive 45–70 kHz USVs are evident in high-ethanol males.

  • High-ethanol males exhibit decreased play attacks and increased anxiety-like behavior.

Abstract

Juvenile rats display rough-and-tumble playing with conspecifics (play fighting behavior) and produce 22 and 50 kHz ultrasonic vocalizations (USVs). The 22 kHz USV is considered to reflect negative emotionality such as anxiety, fear, and distress, whereas the 50 kHz USV is considered to reflect positive emotionality such as joy, happiness, and satisfaction. USV is a sensitive tool for measuring emotionality in socially interactive situations. However, effects of prenatal ethanol-exposure on the acoustic characteristics of play fighting-induced USVs have remained unclear. In Experiment I, we recorded USVs produced by prenatally ethanol-exposed rats during play fighting on postnatal days (PNDs) 40–42 and examined the acoustic characteristics of negative and positive emotion-induced USVs. In Experiment II, we examined the anxiety levels through elevated plus maze testing on PNDs 37–39 and frequencies of playful attacks on PNDs 43–45 in ethanol-exposed rats. Ethanol was administered to pregnant rats in three gradually increased concentrations between gestational days (GDs) 8 and 20. From GDs 14 to 20, ethanol-containing tap water at concentrations of 30% and 15% (v/v) was administered to the high- and low-ethanol groups, respectively. Tap water without added ethanol was given to the control group. On PNDs 40–42, three rats from the same sex and same ethanol concentration group but from different litters were placed together into a playing cage for play fighting. The high-ethanol male triads displayed elevations of 20–35 kHz USVs reflecting negative emotionality and reductions of 45–70 kHz USVs reflecting positive emotionality compared with both the low-ethanol and control male triads. The high-ethanol male triads had prominent elevations of 20–35 kHz USVs with durations longer than 200 ms, whereas the control male triads did not produce such 20–35 kHz USVs at all. There was no difference in USV acoustic characteristics among the female triads. In addition, the high-ethanol male rats exhibited greater anxiety levels and less frequencies of play fighting than the control male rats. Altogether, we conclude that prenatal exposure to ethanol enhances negative emotionality such as anxiety and, accordingly, 20–35 kHz USVs reflecting negative emotionality are produced with a marked decrease in play fighting, suggesting difficulties in social interactions with conspecifics.

Introduction

Alcohol is one of the most widely used recreational drugs worldwide. In low doses, alcohol consumption can reduce anxiety and render people euphoric, cheerful, and more sociable. However, higher doses of alcohol consumption cause serious health problems including damages to the central and peripheral nervous systems, alcohol dependency, and Korsakoff's syndrome. Among the most dreadful effects due to alcohol consumption is fetal alcohol syndrome (FAS), which is a permanent birth defect syndrome caused by maternal alcohol consumption during pregnancy. FAS is characterized by prenatal or postnatal growth deficiency, central nervous system damage/dysfunction, and a unique cluster of facial anomalies (Jones et al., 1973; Astley & Clarren, 2000; Hoyme et al., 2005). Exposure to alcohol during development produces a spectrum of behavioral and cognitive deficits grouped under fetal alcohol spectrum disorders (FASD) (Riley & McGee, 2005; Manning & Hoyme, 2007). Cognitive deficits due to alcohol exposure during development include mental retardation and difficulties in learning, memorizing, problem-solving, and maintaining attention (Streissguth et al., 1991; Ripabelli et al., 2006). Moreover, humans with FASD also display difficulties in understanding social interactions, including difficulties in interpersonal relationships (Thomas et al., 1998), abnormal play behavior (Jacobson et al., 1993), and insecure attachment (O’Connor et al., 1992).

In recent years, research has revealed the importance of play for the neural and behavioral development of an individual to equip him/her adequately for the needs in adulthood (Vanderschuren et al., 1997). Social play deficits can lead to various physical and mental health problems including childhood obesity and attention-deficit hyperactivity disorder as revealed in recent studies (Panksepp, 2007; Burghardt, 2010). Previous studies on the most severe human form of prenatal alcohol exposure indicate that juveniles and adults suffering from FAS or fetal alcohol effects show a lack of reciprocal friendship, lack of tact, and difficulties in cooperating with peers (Streissguth et al., 1991; LaDue et al., 1992). Brown et al. (1991) found no differences in free play behaviors or aggression levels over a 5 min observation period in children exposed to moderate amounts of alcohol during their fetal development. But in the same population, teachers reported more aggressive behaviors and other externalizing problems in children whose mothers had consumed alcohol throughout the pregnancy compared with children whose mothers had either stopped drinking in the third trimester of pregnancy or had no history of drinking during pregnancy (Brown et al., 1991). Steinhausen (1996) examined children with FAS who were exposed to large amounts of alcohol during fetal development. Severe deficits were reported both in teachers' and parents' behavior ratings showing that the social problems were almost as pervasive as attention problems. Thomas et al. (1998) found that children with FAS had deficits in social skills of approximately three standard deviations below the mean value for their age. In both interpersonal skills and use of play and leisure time sub domains of the Vineland Adaptive Behavior Scale, alcohol-exposed children showed significantly greater deficits than the control group. Thus, alcohol consumption by mothers during pregnancy threatens the normal development of social behavior in children.

However, human studies are often complicated by co-ingestion of other substances such as tobacco, marijuana, or psychotropic drugs and other factors like nutritional status, prenatal care, socioeconomic status, genetic variability that cannot be controlled outside clinical studies and those would not be ethical. Animal model systems play an important role to examine the effects of prenatal ethanol exposure on social behavior, allowing strict control of factors such as genetic variation, nutritional status, and drug use as well as timing, dose, and duration of ethanol exposure.

In animal model studies, behavioral alterations that follow prenatal ethanol exposure have been reported for a number of social activities, including play fighting (Meyer & Riley, 1986; Royalty, 1990; Lawrence et al., 2008), mating (Gass et al., 2007), and aggressive behavior during social interactions (Lugo et al., 2006). Despite differences in the timing, doses, and duration of ethanol exposure, as well as sex and age of the animal at which social behavior was measured, these studies consistently reveal ethanol-induced alterations in social behavior during the juvenile period and into adulthood. Studies in which animals were exposed to high doses of ethanol throughout the entire gestational (4.5 g/kg ethanol) and during the early postnatal periods (3.0 g/kg ethanol) demonstrate an increase in play fighting behavior during the juvenile period along with alterations in the somatosensory cortex activity associated with play behavior (Lawrence et al., 2008). Enhanced play fighting behavior both in juvenile male and female rats has also been reported by Royalty (1990) after a moderate level of maternal ethanol exposure (2.0 g/kg) during a shorter period. Prenatal exposure to a low concentration (5%) of ethanol also increased the frequency of play interactions (Hamilton et al., 2010). In contrast, ethanol exposure from gestational days (GDs) 6 to 21 lowered play behavior in rats regardless of age or sex as assessed in the social interaction test at postnatal day (PND) 28 or 42 (Wellmann & Mooney, 2015). Interestingly, maternal ethanol exposure during gestation was found to decrease play fighting behavior in juvenile males but increase play in their female counterparts (Meyer & Riley, 1986). Juvenile rats prenatally exposed to an acute dose of ethanol on GD 7 showed decreased play fighting behavior and social motivation, whereas changing the timing of exposure to GD 12 decreased play fighting behavior in males only (Mooney & Varlinskaya, 2011). Thus, timing, dose, and duration of the ethanol exposure define the way in which ethanol alters social play interactions during the juvenile period.

Ultrasonic vocalizations (USVs) emitted by rats are considered reflecting their real-time emotional state and are widely accepted as animal models of affect in social interactions (Burgdorf et al., 2008; Brudzynski, 2013). Rats produce USVs in the 22–28 kHz and 50–55 kHz frequency ranges indicative of their negative and positive emotional states, respectively (Burgdorf et al., 2008). Rats emit 50 kHz USVs during rough-and-tumble play, mating, or when tickled playfully by a skilled experimenter (Panksepp & Burgdorf, 2000; Knutson et al., 2002). Recent findings suggest that juvenile rats are using vocalizations as play signals (Himmler et al., 2014; Kisko et al., 2015). Vocalizations emitted by juvenile rats are mostly in the 50–55 kHz frequency range associated with a positive emotional state (Knutson et al., 1998; Burgdorf et al., 2008). In particular, 50 kHz vocalizations occur more frequently prior to the play fighting and rarely immediately after the fighting (Himmler et al., 2014). Juvenile rats start their play fighting with original high levels of 50 kHz USVs that decline over time, as negative emotion-induced 22 kHz USVs become more prominent later in the play session (Knutson et al., 2002) when subordinates are defeated by the dominant play partners.

Rodent models of FASD show alterations in social behavior (Marino et al., 2002; Lugo et al., 2006; Hamilton et al., 2014; Wellmann et al., 2015) and communication in the form of USVs (Zimmerberg & McDonald, 1996; Marino et al., 2002; Wellmann et al., 2015). Prenatal exposure to ethanol increases the latencies to emit 50 kHz USVs when a social partner is introduced during social play. Moreover, animals exposed to ethanol prenatally vocalize less and exhibit less play fighting than control-treated animals (Wellmann et al., 2015). In another study (Thakore et al., 2016), high-concentration exposure to ethanol (15% and 30%) enhanced the 22–28 kHz USV emissions in male rats. USV acoustic parameters (i.e., mean frequency in kHz) of both negative and positive USVs were significantly decreased in male rats chronically drinking higher concentrations of ethanol (15% and 30%). Waddell et al. (2016) reported that prenatal ethanol exposure initiates fewer bouts of play fighting behavior during a social interaction test in the juvenile period and decreases the emission of positive emotion-induced 50 kHz USVs. Initiation of play at PND 28 was decreased in both males and females due to ethanol. This effect was also apparent at PND 42 in males only.

As mentioned above, prenatal ethanol exposure produces alterations in play fighting behavior in rats during their juvenile period. Nevertheless, the effects of prenatal ethanol exposure on acoustic characteristics of play fighting-induced USVs in juvenile rats remain to be determined in details. The aim of Experiment I was to clarify the acoustic characteristics of negative and positive emotion-induced USVs in juvenile play fighting. We predicted that prenatal ethanol exposure increases negative emotion induced-USVs and decreases positive emotion-induced USVs.

Section snippets

Experiment I: Effects of prenatal ethanol exposure on negative and positive emotion-induced ultrasonic vocalizations in play fighting

In Experiment I, play fighting-induced USVs were recorded from prenatally ethanol exposed juvenile rats. We classified USVs into two categories reflecting negative and positive emotionality and clarified the acoustic characteristics of these two categories of USVs.

Experiment II: Effects of prenatal ethanol exposure on play fighting and anxiety-like behaviors

Experiment I demonstrated that high ethanol exposure increases the negative emotion-induced USVs and decreases the positive emotion-induced USVs in male play fighting triads only. These results suggest the reduction of play fighting frequencies and elevation of negative emotionality in the high-ethanol male rats. In Experiment II, we recorded the play fighting and anxiety-like behaviors in the high-ethanol male rats and compared with those of the control male rats. We predicted lower

Prenatal ethanol exposure and negative emotionality

Social play or play fighting is the most common form of play in juvenile rats, facilitating social organization in a group or between partners to express and understand intraspecific communicative signals (Meaney & Stewart, 1981; Vanderschuren et al., 1997). The most common scenario in play fighting is for one partner to gain an advantage over the other usually by contacting the nape of his/her neck (Pellis & McKenna, 1995; Pellis et al., 1996). This dorsal contact can motivate the contacted

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by the Graduate Grant Program of Graduate School of Humanities and Human Sciences, Hokkaido University, Japan.

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