RESEARCH ARTICLE

Auditory perception exhibits sexual dimorphism and lefttelencephalic dominance in Xenopus laevisYanzhu Fan1,2, Xizi Yue1, Fei Xue3, Jianguo Cui1,*, Steven E. Brauth4, Yezhong Tang1 and Guangzhan Fang1,*

ABSTRACTSex differences in both vocalization and auditory processing havebeen commonly found in vocal animals, although the underlyingneural mechanisms associated with sexual dimorphism of auditoryprocessing are not well understood. In this study we investigatedwhether auditory perception exhibits sexual dimorphism in Xenopuslaevis. To do this we measured event-related potentials (ERPs)evoked by white noise (WN) and conspecific calls in thetelencephalon, diencephalon and mesencephalon respectively.Results showed that (1) the N1 amplitudes evoked in the righttelencephalon and right diencephalon of males by WN aresignificantly different from those evoked in females; (2) in males theN1 amplitudes evoked by conspecific calls are significantly differentfrom those evoked by WN; (3) in females the N1 amplitude for the leftmesencephalon was significantly lower than for other brain areas,while the P2 and P3 amplitudes for the right mesencephalon were thesmallest; in contrast these amplitudes for the left mesencephalonwere the smallest inmales. These results suggest auditory perceptionis sexually dimorphic. Moreover, the amplitude of each ERPcomponent (N1, P2 and P3) for the left telencephalon was thelargest in females and/or males, suggesting that left telencephalicdominance exists for auditory perception in Xenopus.

KEY WORDS: Auditory perception, Sexual dimorphism,Telencephalon, Event-related potentials (ERPs), Xenopus laevis

INTRODUCTIONDistinct vocalizations are often used to convey multiple types ofinformation about species, sex, reproductive status and location invocal species including birds, insects and anuran amphibians(Gerhardt and Bee, 2006; Naguib et al., 2009; Tobias et al., 2010;Xu et al., 1996). Furthermore, vocal communication, including theproduction, transmission, perception and responses to these acousticsignals, is essential for both survival and reproductive success inthese species (Kelley, 2004; Naguib et al., 2009; Vignal and Kelley,

2007). Accordingly, communication sounds typically exhibitdistinctive spectral and/or temporal attributes that may varyamong individuals or between sexes thus providing sources ofinformation for the neural processes underlying speciesdiscrimination and individual recognition. Consistent with this,species employing vocal communication typically exhibit sexualdimorphism in their responses to conspecific or heterogenousvocalizations at the behavioral, electrophysiological and geneexpression levels (Avey et al., 2008; Bernal et al., 2007; Pollack,1982; Williams, 1985).

For example, male swamp sparrows (Melospiza georgiana)respond aggressively to synthetic songs made up of naturallyoccurring swamp sparrow syllables, even if these are presented inan uncharacteristic temporal sequence of swamp sparrows (Peterset al., 1980; Searcy et al., 1981a). Female swamp sparrows,however, preferentially respond with a soliciting display to songsmade up of swamp sparrow syllables arranged in the species-specific temporal pattern (Searcy et al., 1981b). Similarly, anuranmales are much more likely than females to respond to species-typical signals which show marked variation (Bernal et al., 2007).Moreover different call note types have been shown to conveyseparate messages to males and females in some anuran species(Wells and Schwartz, 2007). At the neural level, the significance ofacoustic features can differ for males and females accompanied bysex differences in the sensitivity of neurons in the auditoryperiphery and midbrain in anurans (Narins and Capranica, 1976;Shen et al., 2011). For example, the sexes may differ in thefrequency at which they are most sensitive, particularly at the highbest frequency (HBF) of the basilar papilla (BP) (Keddy-Hectoret al., 1992; McClelland et al., 1997; Narins and Capranica, 1976;Wilczynski et al., 1984). In addition, call stimulation inducessignificantly higher neuronal expression of the transcription factorZENK compared to silence in the hippocampus and auditoryforebrain areas (i.e. the caudomedial nidopallium and mesopallium)of female but not male zebra finch (Taeniopygia guttata) (Gobeset al., 2009). These results support the idea that male and femalezebra finches show different patterns of neuronal activation inresponse to sexually dimorphic calls. Nevertheless, the prevalenceof such sex differences in auditory processing and perception acrossland vertebrate species remains unclear. The present study focusedon an anuran species, Xenopus laevis, insofar as all land vertebratesare derived from an amphibian stem.

In anurans, survival and reproductive behaviors depend primarilyon a listener’s ability to parse incoming sound signals that conveyspecies identity and reproductive state (Bee, 2012). Anuranstypically exhibit a small vocal repertoire and communicate inwell-defined behavioral contexts making these species well suitedfor studies of acoustic signal perception and auditory systemprocessing (Mangiamele and Burmeister, 2008; Mudry et al., 1977).For most anuran species, social behavior is generally characterizedby sex differences in the production of vocalizations (Diekamp andReceived 24 May 2018; Accepted 12 October 2018

1Department of Herpetology, Chengdu Institute of Biology, Chinese Academy ofSciences, No.9 Section 4, Renmin South Road, Chengdu, Sichuan, People’sRepublic of China. 2University of Chinese Academy of Sciences, 19A YuquanRoad, Beijing, People’s Republic of China. 3Sichuan Key Laboratory ofConservation Biology for Endangered Wildlife, Chengdu Research Base of GiantPanda Breeding, 26 Panda Road, Northern Suburb, Chengdu, Sichuan 610081,People’s Republic of China. 4Department of Psychology, University of Maryland,College Park, MD20742, USA.

*Authors for correspondence (cuijg@cib.ac.cn; fanggz@cib.ac.cn)

X.Y., 0000-0002-7037-2868; F.X., 0000-0001-8563-6793; J.C., 0000-0001-8746-2803; G.F., 0000-0003-1803-6610

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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Gerhardt, 1992). Under the classic paradigm, males are highly vocaland generally produce species-specific advertisement calls to attractfemales for breeding, as well as to deter rivals (Arch and Narins,2009; Kelley, 2004; Tobias et al., 2010). In addition, females areoften mute or possess a severely limited vocal repertoire withlimited complexity (McClelland andWilczynski, 1989; McClellandet al., 1997; Stewart and Rand, 1991). In some anuran speciesauditory tuning characteristics exhibit sexual dimorphism (Hallet al., 2016; Narins and Capranica, 1976; Wilczynski, 1986;Wilczynski and Capranica, 1984;Wilczynski et al., 1992), implyingthat sex differences could well exist in auditory perception in thesespecies. However, very little is known or has been hypothesizedabout sexual dimorphism in central nervous system auditoryprocessing.The South African clawed frog, X. laevis, is a totally aquatic

nocturnal species inhabiting silt-filled ponds that exhibits arelatively rich vocal repertoire (Tinsley and Kobel, 1996; Tobiaset al., 2010; Vignal and Kelley, 2007). The frogs produce sexuallydistinctive calls based on simple click trains (trills) which differ inrate, temporal structure and intensity modulation for socialcommunication (Brahic and Kelley, 2003; Rhodes et al., 2007).Xenopus males can produce six call types, the most prominent ofwhich are advertisement calls produced during the breeding seasonto attract gravid females and compete with rivals (Mangiamele andBurmeister, 2008; Tobias et al., 2004). Xenopus laevisadvertisement calls exhibit a bimodal temporal pattern consistingof fast (19 ms inter-click intervals) and slow (38 ms inter-clickintervals) trill portions chained together in repetitive bouts (Elliottet al., 2007; Tobias et al., 2010), although it is still unclear whichportion (the fast trill or slow trill) constitutes the initial period of thecalls. Xenopus laevis females produce two types of calls, a sexuallyreceptive rapping call that increases male vocal activity and anunreceptive ticking call that depresses male vocal activity (Tobiaset al., 1998). For reproductive success both females and males mustdistinguish among these different signal variants (Andersson andSimmons, 2006; Vélez et al., 2013). Previous studies have showedthat each male and female call type is distinguished by characteristicclick rates (Vignal and Kelley, 2007). Thus males can use click rateto distinguish the sex of callers and discriminate between the femaleticking and rapping calls (Elliott and Kelley, 2007; Vignal andKelley, 2007). Consequently, click rate in particular plays a primaryrole for vocal communication in X. laevis (Elliott et al., 2011).In anurans including X. laevis, auditory information derived from

the tympanum is conveyed by the VIIIth nerve, which projects to thedorsal medullary nucleus (Edwards and Kelley, 2001; Elliott et al.,2011), which then projects to the laminar nucleus of the midbraintorus semicircularis (TS) (Edwards and Kelley, 2001; Kelley, 1980).From the midbrain, ascending auditory signals are conveyed to thecentral amygdala (CeA) in the telencephalon via the centralthalamic nucleus (Hall et al., 2013). Both the TS and CeA areimportant brain areas for the temporal processing of auditory signalsin X. laevis (Edwards and Kelley, 2001; Hall et al., 2013). Previousstudies have demonstrated a high degree of sexual dimorphism forthe central nervous system (CNS) vocal pathways, the larynx itselfand the sensitivity of the peripheral auditory apparatus to species-specific frequencies in male advertisement calls in X. laevis (Hallet al., 2016; Kelley, 1986). For this reason we hypothesized thatCNS processing for auditory perception is also sexually dimorphicin this species. Furthermore, it is well established that discrete brainregions are specialized for different functions (Kandel et al., 2013)and important neuroanatomical features of the brain have beenconserved during vertebrate brain evolution (Finlay et al., 2001;

Northcutt, 2002). Although the amphibian forebrain is not as welldifferentiated as that of amniotes (Feng and Schellart, 1999;Wilczynski, 1992; Wilczynski and Endepols, 2007), the striatumand the superficial and deep thalamic structures have beenimplicated in call recognition (Endepols et al., 2003) andelectrophysiological studies have shown that communicationsounds are processed preferentially in the left hemisphere in Emeimusic frogs (Babina daunchina) (Fang et al., 2014). Accordingly,we also hypothesized that left telencephalic dominance for auditoryperception exists in X. laevis.

Event-related potentials (ERPs) are voltage fluctuations in theelectroencephalogram (EEG) induced within the brain that are timelocked to sensory, motor or cognitive events (Friedman et al., 2001).In this study ERPs were used to assess auditory processing inX. laevis, in midbrain and forebrain structures. To do this werecorded the EEG from telencephalic, diencephalic andmesencephalic sites and measured changes in three ERPcomponents (N1, P2 and P3) evoked by three different stimuli,fast-slow trill calls (FS), slow-fast trill calls (SF) and white noise(WN), a stimulus lacking any of the temporal and spectralcharacteristics of the sounds produced by this species. AuditoryERPs provide a rich source of information about CNS informationprocessing in structures activated by auditory stimuli, and researchhas shown that specific ERP components reflect specific aspects ofauditory perception such as attention, stimulus categorization andthe recognition of stimulus novelty (Näätänen and Winkler, 1999).Auditory ERPs are generally composed of three main components(N1, P2 and P3) which peak at latencies of ∼80 ms, ∼200 ms and∼300 ms, respectively (Luck and Kappenman, 2011; Näätänen andPicton, 1987; Shahin et al., 2005; Tremblay et al., 2001; Tremblayand Kraus, 2002). Functionally, N1 is sensitive to selective attention(Näätänen and Picton, 1987), P2 reflects neural processes sensitiveto the subject’s familiarity with the acoustic stimulus and thespecific acoustic features required to evaluate and classify stimulipreviously experienced (Shahin et al., 2005), while P3 reflects brainactivity corresponding to the psychological changes induced by thestimulation (Polich, 2011), also known as ‘novelty P300’ (Friedmanet al., 2001). The present study measured the amplitudes andlatencies of each ERP component for the left and right hemispheresin response to three acoustic stimuli (FS, SF andWN) for both sexesin order to investigate whether auditory perception is sexuallydimorphic and to determine if left telencephalic dominance exists.

RESULTSThe grand average of the original waveforms includingN1, P2 and P3are shown for each stimulus and each brain region in Fig. 1. Therewere significant differences among stimuli, brain areas and sexes inamplitude rather than latency for each ERP component, respectively.

N1The analysis for the N1 amplitude showed that there was nosignificant main effect for the factors ‘sex’ (F1,14=0.782, partialη2=0.053, P=0.391), ‘stimulus’ (F2,28=1.529, ε=0.968, partialη2=0.098, P=0.234) and ‘brain area’ (F5,70=1.031, ε=0.520,partial η2=0.069, P=0.383). However, the interaction among thesethree factors was significant (F10,140=2.118, partial η2=0.131,P=0.027). Simple and simple-simple effects analysis showed thatthe N1 amplitude in the left mesencephalon for females wassignificantly lower than those in the both sides of telencephalon anddiencephalon (F5,35=3.799, ε=0.708, partial η2=0.352, P=0.007;Table 1 and Fig. 2A). The N1 amplitude evoked by WN for maleswas significantly higher than those for females (F1,14=8.423, partial

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η2=0.376, P=0.012; Table 1), particularly in the right telencephalon(t14=3.098, Cohen’s d=1.656, P=0.008) and the right diencephalon(t14=2.731, Cohen’s d=1.460, P=0.016). For males, the N1amplitude evoked by WN was significantly larger than thoseevoked by conspecific advertisement calls (FS and SF calls) in both

sides of telencephalon (F2,14=8.801, ε=0.769, partial η2=0.557,

P=0.003 for the left telencephalon; F2,14=6.818, ε=0.777, partialη2=0.493, P=0.009 for the right telencephalon) and the rightdiencephalon (F2,14=5.426, ε=0.887, partial η

2=0.437, P=0.018)(Table 1 and Fig. 2B).

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Fig. 1. Grand average waveforms for different brain regions during playbacks of WN, FS and SF calls, respectively (n=16). Abbreviations: LT and RT,the left and right telencephalon; LD and RD, the left and right diencephalon; LM and RM, the left and right mesencephalon; WN, white noise; FS, fast-slowtrill call; SF, slow-fast trill call.

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In addition, for N1 latency therewas no significant main effect forthe factors ‘sex’ (F1,14=0.001, partial η2=0.000, P=0.981),‘stimulus’ (F2,28=1.624, ε=0.776, partial η

2=0.104, P=0.215) and‘brain area’ (F5,70=2.193, ε=0.707, partial η2=0.135, P=0.065),however, the interaction between ‘sex’ and ‘brain area’ wassignificant (F5,70=3.125, partial η2=0.182, P=0.013). Simpleeffects analysis showed that the N1 latency in the leftdiencephalon for females was significantly longer than that ofmales (t14=2.361, Cohen’s d=1.181, P=0.033). For males, thelatency in the right mesencephalon was significantly longer thanthose in the left telencephalon and the right diencephalon(F5,35=3.003, ε=0.486, partial η

2=0.300, P=0.023).

P2The analysis for P2 amplitude showed that there was a significantmain effect for the factor ‘brain area’ (F5,70=8.976, ε=0.645, partialη2=0.391, P=0.000) but not the factors ‘stimulus’ (F2,28=0.188,ε=0.742, partial η2=0.013, P=0.829) and ‘sex’ (F1,14=0.630, partialη2=0.043, P=0.441). In addition, the interaction between ‘sex’ and‘brain area’ was significant (F5,70=4.430, partial η2=0.240,P=0.001). For females, P2 amplitude in the right mesencephalonwas significantly lower than those in the other brain areas, while theP2 amplitude in the left telencephalon was significantly higher thanthat in the right diencephalon (F5,35=4.882, ε=0.540, partialη2=0.411, P=0.002; Table 1 and Fig. 3A). For males, the P2

Fig. 2. Means and standard deviationsfor the N1 amplitudes evoked by eachacoustic stimulus in each brain regionfor females (A) and males (B),respectively (n=16). Filled stars denotethat there were significant differencesbetween different brain areas or differentacoustic stimuli (P<0.05). Abbreviations:LT and RT, the left and righttelencephalon; LD and RD, the left andright diencephalon; LM and RM, the leftand right mesencephalon; WN, whitenoise; FS, fast-slow trill call; SF, slow-fasttrill call.

Table 1. Results of simple and simple-simple effects analysis for the amplitudes of N1/P2/P3 as a function of the factors ‘stimulus’, ‘sex’ and‘brain area’

F/t ε Partial η2/Cohen’s d P LSD/t-test

N1Brain area|female F (5,35)=3.799 0.708 0.352 0.007* LT, RT, LD, RD>LMSex|WN F (1,14)=8.423 NA 0.376 0.012* Male>femaleSex|(RT, WN) t14=3.098 NA 1.656 0.008* Male>femaleSex|(RD, WN) t 14=2.731 NA 1.460 0.016* Male>femaleStimulus|(male, LT) F (2,14)=8.801 0.769 0.557 0.003* WN>FS, SFStimulus|(male, RT) F (2,14)=6.818 0.777 0.493 0.009* WN>FS, SFStimulus|(male, RD) F (2,14)=5.426 0.887 0.437 0.018* WN>FS, SF

P2Brain area|female F (5,35)=4.882 0.540 0.411 0.002* LT, RT, LD, RD, LM>RM; LT>RDBrain area|male F (5,35)=8.612 0.525 0.552 0.000** LT, RT, LD, RD, RM>LM; LT>LD

P3Brain area|female F (5,35)=5.450 0.516 0.438 0.001* LT, RT, LD, RD, LM>RM; LT>RDBrain area|male F (5,35)=10.604 0.602 0.602 0.000** LT, RT, LD, RD, RM>LM

Note: The symbols ‘>’ denote that the amplitude of a given ERP component for the brain areas or the gender on the left side of ‘>’ are significantly larger thanthose on the right side, and no significant difference exists among the corresponding conditions on the same side of ‘>’ for each case. The symbols ‘ | ’ denotethe conditions on the left side of ‘ | ’ under the conditions on the right side of ‘ | ’. Abbreviations: F, the F value from ANOVA; t, the t value from a t-test; Partialη2, effect size for ANOVA; Cohen’s d, effect size for a t-test; ε, the values of epsilon of the Greenhouse-Geisser correction; LSD, least-significant difference test;LT and RT, the left and right telencephalon; LD and RD, the left and right diencephalon; LM and RM, the left and right mesencephalon; FS: fast-slow trill call;SF: slow-fast trill call; WN: white noise. *P<0.05; **P<0.001.

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amplitude in the left mesencephalon was significantly lower than inthe other brain areas, while the P2 amplitude in the lefttelencephalon was significantly higher than that in the leftdiencephalon (F5,35=8.612, ε=0.525, partial η2=0.552, P=0.000;Table 1 and Fig. 3B).In addition, for P2 latency there was no significant main effect for

the factors ‘sex’ (F1,14=0.848, partial η2=0.057, P=0.373),‘stimulus’ (F2,28=0.810, ε=0.788, partial η2=0.055, P=0.455) and‘brain area’ (F5,70=1.052, ε=0.719, partial η2=0.070, P=0.394,),however, the interaction between ‘sex’ and ‘brain area’ wassignificant (F5,70=2.566, partial η2=0.155, P=0.034). Simpleeffects analysis showed that the latency in the right telencephalonfor females was significantly longer than for males (t14=2.596,Cohen’s d=1.299, P=0.021).

P3The analysis for the P3 amplitude showed that the main effects for thefactors ‘stimulus’ (F2,28=4.908, ε=0.886, partial η2=0.260, P=0.015)and ‘brain area’ (F5,70=10.038, ε=0.670, partial η2=0.418, P=0.000)but not ‘sex’ (F1,14=0.653, partial η2=0.045, P=0.432) weresignificant. The P3 amplitude evoked by WN was significantlyhigher than those evoked by the FS and SF advertisement calls(Fig. 4A). The interaction between ‘brain area’ and ‘sex’ wassignificant (F5,70=5.041, partial η

2=0.265, P=0.001). Simple effectsanalysis showed that the P3 amplitude in the right mesencephalon forfemales was significantly lower than those in the other brain areas.Furthermore, P3 amplitude in the left telencephalon was significantlyhigher than in the right diencephalon (F5,35=5.450, ε=0.516, partialη2=0.438, P=0.001; Table 1 and Fig. 4A). For males, the P3amplitude in the left mesencephalon was significantly lower thanthose in the other brain areas (F5,35=10.604, ε=0.602, partialη2=0.602, P=0.000; Table 1 and Fig. 4B). In addition, for P3latency there was no significant main effect for the factors ‘sex’(F1,14=0.206, partial η

2=0.014, P=0.657), ‘stimulus’ (F2,28=1.427,ε=0.723, partial η2=0.093, P=0.258) and ‘brain area’ (F5,70=1.289,

ε=0.732, partial η2=0.084, P=0.279), and there was no interactionamong these factors.

DISCUSSIONThe present study shows that when three stimuli consisting of WN,FS and SF calls are presented with equal probability (1) theamplitudes of N1 evoked in males by the WN stimulus for the righttelencephalon and right diencephalon are significantly differentfrom those evoked in females; (2) in males the N1 amplitudesevoked by conspecific calls are significantly different from thatevoked by the synthesized WN stimulus while no differenceobtained between FS and SF calls; (3) in females the N1 amplitudefor the left mesencephalon was significantly lower than for otherbrain areas, while the P2 and P3 amplitudes for the rightmesencephalon were the smallest. In contrast these amplitudes forthe left mesencephalon were the smallest in males. These results areconsistent with the hypothesis that neural processing for auditoryperception is sexually dimorphic.

Sexual dimorphism in auditory perceptionIn anurans, sex differences in acoustic communication arewidespread (Hoke et al., 2008; Kelley, 1986, 2004; Liu et al.,2014). At the behavioral level, different types of call notes canconvey separate messages to males and females in some anuranspecies (Wells and Schwartz, 2007). For this reason, males andfemales often react differently in response to conspecific calls. Inaddition, males are much more likely than females to respond tosignals which vary from the species’ norm (Bernal et al., 2007).These sexually dimorphic behaviors depend on neural systems thatare sex-specific or common to males and females and potentiallyregulate a number of behaviors (Hoke et al., 2010). For example,in three Xenopus species (X. amieti, X. petersii and X. laevis),peripheral auditory sensitivity to the two dominant frequencies ofconspecific male advertisement call is enhanced in femalescompared to males, while males are most sensitive to lower

Fig. 3. Means and standard deviationsfor the P2 amplitudes evoked by eachacoustic stimulus in each brain regionfor females (A) and males (B),respectively (n=16). Filled stars and openstars denote that there were significant(P<0.05) and extremely significant(P<0.001) differences between differentbrain areas, respectively. Abbreviations:LT and RT, the left and righttelencephalon; LD and RD, the left andright diencephalon; LM and RM, the leftand right mesencephalon; WN, whitenoise; FS, fast-slow trill call; SF, slow-fasttrill call.

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frequencies including those in the male-directed release calls (Hallet al., 2016). Furthermore, the sensitivity to these spectral features ismodulated by ovarian signals in female X. laevis.In the present study, WN served as a proxy for non-vocal signals

which differ substantially from conspecific signals. N1 amplitudesin the right forebrain evoked by WN were significantly higher inmales than in females. Moreover, N1 amplitude evoked by WNwassignificantly higher in males than those evoked by conspecific calls.These results are consistent with the idea that the negative N1 wavesare affected by selective attention which enhances the perception ofhigh-priority stimuli at the expense of other stimuli in theenvironment (Näätänen and Picton, 1987; Woldorff et al., 1993).Male X. laevis frogs produce advertisement calls to attract mates andcompete with other males in the breeding season (Tobias et al.,2004), while females in the reproductive state are often silent orrespond less to males (Tobias et al., 1998). Thus males would bemore easily detected by predators compared to females. Animalsgenerally maintain alertness to non-conspecific sounds which maybe associated with danger (Haff and Magrath, 2010). Accordinglythis strong selective pressure would likely result in a larger ‘N1effect of selective attention’ (Hillyard et al., 1973) in malescompared to females. Furthermore, for anurans, the sexes do differin sensitivity at audiogram best frequencies, with males moresensitive in the lower frequency range (Hall et al., 2016; Mirandaand Wilczynski, 2009). Females are more sensitive than males inresponse to natural vocalizations, despite showing no difference inresponse to pure tones at the same frequencies found withinadvertisement calls. However, thresholds to frequencies outside therange of the male advertisement call are higher in females (Liu et al.,2014; Miranda and Wilczynski, 2009). In addition, N1 is known tobe sensitive to onset parameters (Biermann and Heil, 2000) and isinfluenced more by synchronous activity induced by the temporalenvelope of the stimuli than by the spectral content in humans(Shahin et al., 2005). Thus, it is reasonable to speculate that

differences between the temporal-spectral characteristics of theonsets of WN and the conspecific calls might contribute to thecurrent results.

Significant differences in N1 amplitudes between sexes inhumans have been found mainly in the right forebrain. Theseresults are consistent with the idea that an important attentionalnetwork including the temporoparietal cortex and inferior frontalcortex is largely lateralized to the right hemisphere and specializedfor the detection of behaviorally relevant stimuli, particularly whenthey are salient or unexpected (Corbetta and Shulman, 2002; Nobreet al., 2004; Shulman et al., 2010). More broadly, the righttelencephalon has been proposed to be the site for rapid control ofattention modulation (Evans et al., 2000; Fang et al., 2015;Yamaguchi et al., 2000), enabling individuals to efficiently acquireor change targets. Thus, right hemisphere dominance for selectiveattention and target detection might be a primitive trait present inamphibia. However it is noteworthy that the present results differfrom our previous study showing a sexually dimorphic lateralizedattention modulation network in Emei music frogs using Grangercausal connectivity analysis, i.e. Granger causal connections in theleft telencephalon are stronger in males while those in the righttelencephalon are stronger in females (Xue et al., 2018). Thisdifference might reflect species differences.

The present results show that the N1 latency for the leftdiencephalon and the P2 latency for the right telencephalon infemales were significantly longer than those in males. Theseresults are consistent with the idea that the latencies of ERPcomponents are always positively correlated with perceptualprocessing demands (Callaway and Halliday, 1982; Courchesneet al., 1985; Kutas et al., 1977; Luck, 2014; Magliero et al., 1984;Polich, 2007) and that females are typically more selective inmate choice due to their greater reproductive investment. Inaddition, in females both P2 and P3 amplitudes for the rightmesencephalon were significantly lower than those for other

Fig. 4. Means and standard deviationsfor the P3 amplitudes evoked by eachacoustic stimulus in each brain regionfor females (A) and males (B),respectively (n=16). Filled stars and openstars denote that there were significant(P<0.05) and highly significant (P<0.001)differences between different brain areas,respectively. Abbreviations: LT and RT,the left and right telencephalon; LD andRD, the left and right diencephalon; LMand RM, the left and rightmesencephalon; WN, white noise; FS,fast-slow trill call; SF, slow-fast trill call.

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brain areas, consistent with the idea that left-hemisphericdominance for conspecific communication sounds exists inmany vertebrates including frogs (Fang et al., 2014; Xue et al.,2015). Surprisingly, in males both P2 and P3 amplitudes for theleft mesencephalon were the lowest. Future research is needed todetermine the cause of this sexual dimorphism.

The left telencephalon may play an important role inauditory perceptionThe present results show that the amplitude of each ERP component(N1, P2 and P3) for the left telencephalon are the largest of those forall other brain areas studied here in females and/or males. The P2component reflects the process of signal evaluation and classification,and its amplitude can be enhanced by familiarity or similaritybetween the target and current stimulus (Bosnyak et al., 2004; Pottset al., 1998; Reinke et al., 2003; Shahin et al., 2005; Tremblay et al.,2010).While the P3 component can be elicited by a variety of stimuli,including those that are defined as targets as well as novels (Friedmanet al., 2001; Novak et al., 1992), P3 amplitude is related to thecognitive processing demands of the eliciting stimulus (Anderer et al.,1998; Woodman, 2012). Thus the present results suggest that the lefttelencephalon plays an important role in auditory perception.In amphibians, the midbrain torus semicircularis serves to relay

brainstem auditory input to the forebrain as well as acting as a centerfor integrating ascending auditory and descending forebrain inputs(Endepols andWalkowiak, 2001; Wilczynski, 1981). The torus alsoacts as an audiomotor interface (Luksch and Walkowiak, 1998).Pathways originating in the midbrain give rise to ascending auditoryinput to the diencephalon and telencephalon, which isextraordinarily widespread. Nearly all of the telencephalon exceptspecific olfactory areas receive some auditory input because theamphibian forebrain is relatively undifferentiated (Feng andSchellart, 1999; Wilczynski, 1992; Wilczynski and Endepols,2007). For this reason there is no specific sensory area in the anurantelencephalon directly comparable to the auditory areas of theamniote telencephalon. Compared to amniotes the anuran pallium isnot parcellated into discrete functional areas, although widespreadconnections linking forebrain neurons to motor and/or endocrinesystems and limbic structures exist (Wilczynski and Endepols,2007). However our knowledge about specific neuronal responseproperties in the anuran telencephalon is limited (Wilczynski andEndepols, 2007).In contrast much is known about the role of the anuran midbrain

in acoustic signal processing including IEG expression in responseto conspecific calls (Burmeister et al., 2008; Wilczynski and Ryan,2010). Studies have also reported IEG expression in multipleforebrain areas in response to conspecific calls (Hoke et al., 2007,2010; Mangiamele and Burmeister, 2008). Ascending toralefferents gather in the lateral rostral torus and project to theipsilateral diencephalon which consists of the thalamus dorsally andhypothalamus ventrally (Butler, 1995; Wilczynski and Endepols,2007). Neurons in the posterior thalamic nucleus appear to bespecialized for processing the temporal and spectral features of thespecies vocalizations, particularly advertisement calls (Wilczynski,1992; Wilczynski and Endepols, 2007).Previous studies have shown that two telencephalic areas, the

striatum and medial pallium, receive ascending auditory input fromthe thalamus and contain neurons responsive to acoustic stimuli(Wilczynski and Endepols, 2007; Wilczynski and Ryan, 2010).Simple stimuli such as clicks generally fail to stimulate cells at thislevel of the CNS. In contrast, complex signals similar to naturallyoccurring calls can evoke large neuronal responses in the striatum

and medial pallium (Wilczynski and Capranica, 1984). Moreover,lesions of the striatum and superficial and deep thalamic structuresmay disrupt vocal recognition (Endepols et al., 2003), suggestingthat telencephalic areas play important roles in call recognition.Furthermore, we have previously shown that communication soundsare processed preferentially in the left hemisphere in Emei musicfrogs (Fang et al., 2014). Taken together with the present results inX. laevis, the accumulating evidence points to an important role forthe left telencephalon in acoustic signal processing and auditoryperception in anurans.

The present results show that the P3 amplitude evoked by WNwas greater than the P3 amplitudes evoked by both conspecific calltypes, consistent with our previous finding showing that Emeimusic frogs classify conspecific calls into one category, andperceive WN as a novel stimulus (Fang et al., 2015). Typically theP3 component is most strongly elicited using oddball paradigms inwhich subjects are exposed to minor portions of unexpected/novelstimuli interspersed between major portions of uniform/standardstimuli (Friedman et al., 2001; Novak et al., 1992). However, theequiprobability paradigm used in the present study could produce anoddball-like paradigm in which the conspecific FS and SF callscould serve as the major stimulation (66.7% probability) while WNserved as a minor novelty (33.3% probability). Thus, a larger P3could have been evoked by WN because the animals, as would beexpected, classified the two conspecific calls into one category.

MATERIALS AND METHODSAnimalExperiments were performed on sixteen X. laevis frogs of both sexes (eightmales and eight females) bred in our lab. The frogs were separated by sexand housed in two aquaria (120×50 cm and 60 cm deep) with a water depthof approximately 20 cm. The frogs were fed every three days and the waterwas replaced once a week. The aquaria were placed in a room undercontrolled temperature conditions (20±1°C) and maintained on a 12 h:12 hlight–dark cycle (lights on at 08:00). The subjects measured 8.1±1.1 cm(mean±s.d.) in body length and 67.1±22.2 g in body mass at the time ofsurgery. All experimental procedures conformed to the requirements of theAnimal Care and Use Committee of Chengdu Institute of Biology, ChineseAcademy of Sciences.

SurgeryAll experiments were conducted during April to August, 2016 and June,2017 (this species breeds between April and September in our lab), duringwhich time calling in both sexes and a female oviposit were observed.Sexually mature adults of both sexes (males with nuptial pads, females withprotruding cloacal labia) were chosen for the experiments (Hayes et al.,2010; Tobias et al., 2004; Van Wyk et al., 2003). The reproductive status ofmales was determined by recording call activity before and during theexperiments and that of the females was determined by determining if eachfemale was gravid after the experiments.

Before surgery, the frogs were deeply anesthetized by immersion in a0.35% solution of tricaine methanesulfonate (MS-222) and the optimumdepth of anesthesia for surgery was determined by loss of the toe pinchresponse. After anesthesia, the frogs were covered with moist gauze toprevent dehydration. The skin and the underlying muscles of the operationarea were cut away in order to expose the dorsal skull. Seven cortical EEGelectrodes, consisting of miniature stainless steel screws (ϕ 0.8 mm), werescrewed by turning through 3.5 circles to implant (about 1.1 mm deep) in thefrog skull above the left and right sides of the telencephalon, diencephalonand mesencephalon (LT, RT, LD, RD, LM and RM). These electrodes werereferenced to the electrode above the cerebellum (C) (Fig. 5). Ten seconds oftypical EEG waves are presented along with the corresponding electrodepairs in Fig. 5. The electrodes above LT and RT were implanted bilaterally6.4 mm anterior to the lambda (i.e. the vertex where the skull suturesintersect) and 1.0 mm lateral to the midline respectively, and the electrodes

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above LD and RD were implanted bilaterally 3.4 mm anterior to the lambdaand 1.0 mm lateral to the midline respectively, while the electrodes aboveLM and RM were implanted bilaterally 1.4 mm anterior to the lambda and1.0 mm lateral to the midline, respectively. The reference electrode (C) wasimplanted 1.0 mm posterior to the lambda at the midline (Fig. 5). One end ofall electrode leads, formvar-insulated nichrome wires, was twined tightly onthe screws and fixed on the skull of the frog with dental acrylic, while theother end was soldered to the pins of the light connector. If a frog regainedmotility during surgery, supplemental MS-222 solution was wiped onto theanimal’s skin using a cotton swab. Finally, the skin edges and musclessurrounding the wound were treated with an ointment with triple antibioticsand pain relief (CVS pharmacy, Woonsocket, USA) to prevent infection anddiscomfort. In addition, the connector was covered with self-sealingmembrane (Parafilm®; Chicago, USA) for waterproofing.

After surgery, the frogs were covered with moist gauze and were placedon a sponge (17×10×1 cm3) which had absorbed about 200 ml of water. Thespongewas placed at the bottom of a transparent plastic box with a floor areaof 18×11 cm2 and 12 cm in height. After about 3 h, the frogs were recoveredfrom anesthesia status and each frog was housed individually in an aquarium(34×24.5×18.5 cm3, with water depth approximately 10 cm) for 6 days forrecovery before the experiments were performed. During the recoveryperiod, the frog was fed every 3 days and the water replaced every day. Afterthe end of all experiments, the subjects were euthanized by immersion inMS-222 solution for a prolonged period of time and peripheral bloodleukocyte counts were acquired to verify that the animals were not infectedduring the experimental period (Hadji-Azimi et al., 1987) (Fig. S1;Table S1). Finally, the electrode locations were confirmed by injectinghematoxylin dye through the skull holes in which the electrodes hadpreviously been installed (Fig. S2). In addition, the resistances between thereference and each of other electrodes were measured when the subjectswere placed under water, while the corresponding resistances were alsomeasured for a simulative electrode array under water in which the distancesbetween every two electrodes were the same as those in the subjects. Sincethe resistances for the subjects were about 100 times as large as for thesimulative electrode array, possible leakage water around the electrodeswould be slight and could not influence the present results.

Recording conditionsAnaquarium (120×50 cmand 60 cmdeep)withwater depth of approximately20 cm was placed in a soundproof and electromagnetically shielded chamberfor which the background noise was 24.3±0.7 dB (mean±s.d.). In order todecrease sound reverberations which might be produced within the glass, theinner walls of the aquarium were covered by sound-absorbing cotton. Theaquarium was separated into three equal parts by fine-mesh gauze. Light andtemperature in the chamber were maintained as in the housing room. A video

camera with an infrared light source and motion detector was appendedcentrally about 1 m above the middle part of the aquarium for monitoring thesubject’s behaviors. Electrophysiological signals were recorded with a signalacquisition system (Chengyi, RM6280C; Sichuan, China).

Stimuli and procedureThe advertisement calls of X. laevis are biphasic and composed of alternatingfast and slow trills (Elliott et al., 2007; Tobias et al., 2010). Previous work hasshown that white noise (WN) removes the variability of stimulus novelty andcan produce reliable ERP components (Combs and Polich, 2006; Frank et al.,2012; Yue et al., 2017). Accordingly, three types of stimuli were used: WN,FS (fast-slow trill call) and SF (slow-fast trill call, i.e. the reverse sequence ofthe FS stimulus). Since pseudoreplication may affect the conclusions ofstatistical analyses in ecological, animal behavior and neuroscience studies(Freeberg and Lucas, 2009; Kroodsma et al., 2001; Lazic, 2010; Schank andKoehnle, 2009), the possible effects of pseudoreplication were controlled byusing multiple stimulus examplars in the present study. To do this, fouradvertisement calls of virtually identical durations were acquired from fourdifferent individuals by random selection from our dataset. Each callconsisted of a fast trill and a slow trill (i.e. FS). The reverse versions of the fourcalls (i.e. SF) were acquired using simple cut and paste in AdobeAudition 3.0software (San Jose, California, USA). The duration of the WN stimulusequaled the average duration of the four conspecific calls (about 1 s) and wasshaped with rise and fall time sinusoidal periods of 50 ms (Fig. 6). Each set ofstimuli was played back to four subjects using an equiprobability paradigmvia two Daravoc underwater speakers (frequency response: 0.1–10 kHz; SunPride Inc., Zhejiang, China). The speakers were equidistantly placed at thebilateral sides of the testing aquarium. The root mean square (RMS) intensityof each stimulus was adjusted to be the same as that of a randomly selectedcall recorded in the experimental aquarium.Under these conditions, the soundlevel distribution at the bottom of the middle part of the aquariumwas close toa quasi-free sound field. Furthermore, the subjects could move freely in thispart of the aquarium throughout the experiments. Thus it is highly unlikelythat the tiny differences in the stimulus amplitude across the tank bottomcould have had a significant effect on the ERP measures. Since the influenceof the target stimulus probability on P3 amplitudewouldweaken considerablyunder longer interstimulus intervals (Gonsalvez and Polich, 2002; Polich,1987), the interstimulus interval was set to 1.5 s in the present study, the sameas that used in a previous study on another frog species, the Emei music frog(Fang et al., 2015). For each subject, a total of 300 stimulus presentations witheach stimulus presented 100 times were broadcasted in a random order.Randomization was constrained to prevent more than three stimuli fromwithin the same acoustic category being presented successively.

Many studies have referred to the P3 ERP component as occurring mostlyin oddball paradigms in which subjects are exposed to relatively rare

Fig. 5. Electrode placements and 10 s of typical EEG tracings for each channel. The intersection of the three dashed lines in the head of X. laevisdenotes the lambda (i.e. the vertex where skull sutures intersect). Abbreviations: LT and RT, the left and right telencephalon; LD and RD, the left and rightdiencephalon; LM and RM, the left and right mesencephalon. Image adapted from Fan et al. (2018) licensed under https://creativecommons.org/licenses/by/4.0/legalcode CC-BY 4.0 with permission.

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unexpected/novel stimuli interspersed between major time portionsconsisting of a series of uniform/standard stimuli. It should therefore benoted that the equiprobability paradigm used in the present study couldproduce an oddball-like paradigm in which the conspecific FS and SF callscould serve as the major stimulation (66.7% probability) while WN servedas a minor novelty (33.3% probability). In light of this, it would be expectedthat WN would evoke a larger P3 since the subjects would be expected toclassify the two conspecific calls into one category.

EEG signal acquisition and ERP analysisAfter postoperative recovery for 6 days, the subject was placed in themiddle of the experimental aquarium and connected to the signal acquisitionsystem for about 2 h habituation. The connector was covered with a self-sealing membrane for waterproofing and waterproof adhesive was usedwhen necessary. Then the EEG signal and behavioral data were collectedaccording to the above described auditory stimulation paradigm.

To extract ERP components, EEG recordings were filtered offline using aband-pass filter at 0.25–25 Hz and a notch filter to eliminate possibleinterference at 50 Hz before averaging the stimulus-locked EEG epochs.The EEG signals were divided into epochs with a duration of 700 ms,including a prestimulus baseline of 200 ms. All single EEG trials wereinspected visually and trials with muscle artifacts and electrode drifts wereremoved from all further analysis. Accepted trials were averaged accordingto stimulus types and brain areas within each session.

The auditory ERP component N1 was defined as the mean amplitudeduring latency intervals of 30–130 ms, P2 during intervals of 150–250 msand P3 during intervals of 250–350 ms after stimulus onset (Luck andKappenman, 2011; Näätänen and Picton, 1987; Shahin et al., 2005;Tremblay et al., 2001; Tremblay and Kraus, 2002). The latency wasdetermined by the ‘50 percent area latency measure’ for each ERPcomponent (Luck, 2014), i.e. measuring the area under the curve within thetime windows and finding the time point that divided this area into equalhalves. Both the amplitudes and latencies of the original waveforms weresubjected to further statistical analyses for each ERP component.

Statistical analysesThe normality of the distribution and the homogeneity of variance for ERPvalues were estimated with the Shapiro–Wilk W test and Levene’s test,respectively. The amplitudes of N1, P2 and P3 were statistically analyzedusing a four-way repeated measures ANOVA with the variables of ‘sex’(male/female), ‘stimulus’ (FS/SF/WN), ‘brain area’ (LT/RT/LD/RD/LM/RM) and ‘stimulus set’ (the four stimulus sets). There was no significantmain effect of the last factor for either the amplitude or the latency of eachERP component, consistent with the idea that the results of the presentstatistical analyses could not be affected by pseudoreplication. Thus, all datasets were pooled regardless of ‘stimulus set’ and statistically analyzed usinga three-way repeated measures ANOVA including the first three factors.Both main effects and interactions were examined. For significantANOVAs, data were further analyzed for multiple comparisons using theleast-significant difference (LSD) test or t-test. Simple and simple-simpleeffects analysis were applied when the interaction was significant.Greenhouse-Geisser epsilon (ε) values were employed when theGreenhouse-Geisser correction was necessary. Effect size was determinedwith Cohen’s d for t-tests and partial η2 for ANOVAs (partial η2=0.20 is asmall effect size, 0.50 is a medium effect size and 0.80 is a large effect size).SPSS software (release 21) was utilized for the statistical analysis.A significance level of P<0.05 was used for all comparisons.

AcknowledgementsThe authors gratefully acknowledge all themembers of the Behavioral NeuroscienceGroup for their discussion and help with the experiments reported and the authorsacknowledge the journal PeerJ for the permission to reuse Fig. 5.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: Y.F., Y.T., G.F.; Methodology: J.C., G.F.; Investigation: Y.F., X.Y.,F.X.; Writing - original draft: Y.F.; Writing - review & editing: Y.F., S.E.B., G.F.;Supervision: Y.T.; Project administration: G.F.; Funding acquisition: J.C., G.F.

Fig. 6. Waveforms and spectrograms of the three stimuli for a randomly selected stimulus set. (A) WN, white noise; (B) FS, fast-slow trill call; (C) SF,slow-fast trill call. Since the frequency response of the speaker is 0.1–10 kHz, white noise within the same bandwidth is shown.

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FundingThis work was supported by grants from the National Key Research andDevelopment Program of China [No. 2016YFC0500104] and the National NaturalScience Foundation of China [No. 31672305 and No.31372217 to G. F.,No.31772464 to J. C.] and from Youth Innovation Promotion Association of theChinese Academy of Sciences [2012274], Chinese Academy of Sciences ‘Light ofWest China’ Program and Youth Professor Project of Chengdu Institute of Biology[Y3B3011] to J. C.

Data availabilityThe data that support the findings of this study are available from the correspondingauthors upon reasonable request.

Supplementary informationSupplementary information available online athttp://bio.biologists.org/lookup/doi/10.1242/bio.035956.supplemental

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RESEARCH ARTICLE Biology Open (2018) 7, bio035956. doi:10.1242/bio.035956

BiologyOpen

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