Harmonic and frequency modulated ultrasonic vocalizations reveal differences in conditioned and unconditioned reward processing

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ARTICLE IN PRESSG ModelBR 9505 1–8

Behavioural Brain Research xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

esearch report

armonic and frequency modulated ultrasonic vocalizations revealifferences in conditioned and unconditioned reward processing

rik J. Garcia ∗, Talus J. McCowan, Mary E. Cainansas State University, United States

i g h l i g h t s

Rat novelty and sensation seeking is complex and requires more than one measure.FM and harmonic USVs change differently with repeated tickling.The change in harmonic USVs was positively related with novelty seeking.FM and harmonic USVs can be used to understand attribution of incentive value.

r t i c l e i n f o

rticle history:eceived 24 January 2015eceived in revised form 11 March 2015ccepted 22 March 2015vailable online xxx

eywords:ovelty seekingensation seekingltrasonic vocalizationseterospecific playicklingncentive salience

a b s t r a c t

Novelty and sensation seeking (NSS) and ultrasonic vocalizations (USVs) are both used as measures ofindividual differences in reward sensitivity in rodent models. High responders in the inescapable nov-elty screen have a greater response to low doses of amphetamine and acquire self-administration morerapidly, while the novelty place preference screen is positively correlated with compulsive drug seeking.These screens are uncorrelated and implicated in separate drug abuse models. 50 kHz USVs measure affec-tive state in rats and are evoked by positive stimuli. NSS and USVs are each implicated in drug response,self-administration, and reveal differences in individual behavior, yet their relationship with each otheris not understood. The present study screened rats for their response to novelty and measured USVs ofall call types in response to heterospecific play to determine the relationships between these individualdifference traits. Generally, we hypothesized that 50 kHz USVs would be positively correlated with theNPP screen, and that 22 kHz would be positively correlated with the IEN screen. Results indicate none ofthe screens were correlated indicating they are measuring different individual difference traits. However,examination of the subtypes of USVs indicated harmonic USVs and the novelty place preference were

positively correlated. Harmonic 50 kHz USVs increased in response to reward associated context, sug-gesting animals conditioned to the heterospecific tickle arena and anticipated rewarding stimuli, whileFM only increased in response to tickling. USV subtypes can be used to elucidate differences in attributionof incentive value across conditioned stimuli and receipt of rewarding stimuli. These data provide strongsupport that harmonic and FM USVs can be used to understand reward processing in addition to NSS.

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. Introduction

Humans, rodents and other mammalian species have a tendencyo seek out and explore novel environments and stimuli. In humans,he tendency to be a high novelty and sensation seeker is corre-ated with a variety of maladaptive behaviors such as unprotected

Please cite this article in press as: Garcia EJ, et al. Harmonic and freqconditioned and unconditioned reward processing. Behav Brain Res (2

ex and drug experimentation [1–3]. Therefore, several animalodels have been developed to study the relationship between

ovelty and sensation-seeking (NSS) behavior and maladaptive

∗ Corresponding author. Tel.: +1 720 233 3099; fax: +1 785 532 5401.E-mail address: [email protected] (E.J. Garcia).

ttp://dx.doi.org/10.1016/j.bbr.2015.03.049166-4328/© 2015 Published by Elsevier B.V.

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© 2015 Published by Elsevier B.V.

behavior in rodents. When rodents encounter novel stimuli theyshow an increase in locomotor activity that ceases when novelstimuli become familiar or well explored [4]. Research suggestsexploration of novel stimuli activates regions of the mesolimbicdopamine pathway, indicating the presentation of a novel stimulusor the exploration of a novel stimulus is rewarding to the organism[5,6]. Presentation of novel stimuli including odors, objects, visualor tactile cues can decrease amphetamine self-administration [7,8].

The inescapable novelty screen and novelty place preference

uency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

screen are behavioral measures used to measure the response tonovelty in rodents. The inescapable novelty screen challenges ani-mals with a novel open-field apparatus and locomotor activity ismeasured [4]. Dopamine is thought to mediate the increase in

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ocomotor response because inactivation of dopamine in theucleus accumbens reduces novelty-induced locomotor response9,10]. The 30 min inescapable novelty screen is sufficient toeveal differences in novelty-induced locomotor activity betweenodents, which are subsequently divided into high respondersHR) or low responders (LR). High responding rodents display aariety of behaviors in which they are different than their loweresponding counterparts including elevated locomotor responseo low doses of psychomotor stimulants [11,12], and a faster ratef acquisition of self-administration of low unit doses of psychos-imulants [4,7,13,14]. Interestingly, pharmacological interventionsesulting in the elevation of dopamine in the mesolimbic path-ay result in increased exploratory locomotion providing evidence

hat dopaminergic transmission mediates the locomotor responseo a novel environment and to amphetamine-induced locomotorctivity [9,10].

Similar to the inescapable novelty screen, the novelty place pref-rence screen is used to measure the response to novelty in rodents.owever, unlike the inescapable novelty screen where the novelnvironment is unavoidable, the novelty place preference screeneasures the animal’s choice to engage a novel context. In the nov-

lty place preference screen animals can freely move between aamiliar and novel context. Animals with greater amounts of timen the novel context are classified as high novelty seekers (HNP),nd these HNP animals show a propensity to develop compulsiveocaine self-administration behavior, while low novelty seekersLNP) do not show the same compulsive behaviors [15]. Similar toovelty-induced locomotor activity, elevated dopaminergic activ-

ty has been recorded in the mesolimbic pathway when animalsngage the novel context [16]. This elevation in dopamine in thiseward pathway is transient and is not induced by subsequentntries into the previously novel context, indicating the noveltyspect and not the contextual elements were responsible for thelevation in dopamine.

Despite the novelty screens’ relationships with dopamine andddiction, they are uncorrelated, indicating the inescapable nov-lty screen and the novelty place preference screen are measuringifferent aspects of novelty [14,15,17]. One proposed hypothesisor the observed differences is that the inescapable novelty screennduces a stress response as evidenced by an elevation in cor-icosterone [13], while the novelty place preference screen doesot elevate corticosterone [18]. Further support that these screensre measuring different aspects of novelty is that HR rodents aslassified in the inescapable novelty screen are not necessarilyNP rodents in the novelty place preference screen, with similar

esults with LR rodents and LNP rodents [15]. Although each noveltyehavior is mediated by dopaminergic activity, it is unclear howhese screens correlate with other measures of individual differ-nces. Understanding this relationship will bridge the gap betweenhe NSS screens and elucidate novelty’s complex relationship withddiction-like criteria and understand how novelty is implicated inhe processing of rewards [14,15].

Given that NSS in rats is complex and may encompass morehan one dimension, another measure to understand the affec-ive or motivational response of the rodent in real-time could beeneficial. Ultrasonic vocalizations (USVs) are emitted by rats andice and are reflective of the animals’ motivational and/or affec-

ive state [19,20]. Broadly, USVs can be dichotomized into 2 callypes reflecting positive and negative affective states. Twenty-wo kHz calls serve as alarm calls and are evoked by aversivetimuli including startling air puffs and withdrawal from drugsf abuse [21–23]. These calls are indicative of a negative affec-

Please cite this article in press as: Garcia EJ, et al. Harmonic and freconditioned and unconditioned reward processing. Behav Brain Res (2

ive state. Alternatively, 50 kHz calls are evoked by unconditionedewarding stimuli including psychomotor stimulants [24–31], andickling, termed ‘heterospecific play’ or anticipation of rewards32–35].

PRESSResearch xxx (2015) xxx–xxx

Tickling is a procedure designed to mimic rodent play behaviorand is rewarding to rodents. Rats will readily self-administer tick-ling and not general hand contact [36], and rats readily develop aconditioned place preference for a context paired with tickling [36].50 kHz vocalizations can be used as a measure of the individualincentive value attributed to the reinforcer, with greater numbersof 50 kHz vocalization indicative of a larger perceived magni-tude of the reinforcer. Rodents with greater 50 kHz vocalizationsdemonstrated a greater conditioned place preference [37–39], indi-cating the subjective value of the reinforcer can be measured with50 kHz vocalizations. Pharmacological manipulations of dopamineD1 and D2 receptor function suggest 50 kHz are strongly depend-ent on dopamine function [40]. Therefore; USVs could be used tounderstand the affective state of the rat to elucidate the differentmotivational states of NSS including the aversive and the rewardingelements.

Novel stimuli are thought to be rewarding, yet animal modelsof novelty are uncorrelated. Furthermore, the inescapable noveltyscreen is known to elevate corticosterone and may be a measureof stress-induced locomotor activity rather than novelty-inducedlocomotor activity, while the novelty place preference screen meas-ures the animal’s choice to engage a novel context. The presentstudy examined rat behavioral responses in the inescapable nov-elty screen, novelty place preference screen, and to heterospecificplay, to determine the relationship between NSS and USVs. Wehypothesized the inescapable novelty screen and 22 kHz vocali-zations would be positively correlated because of the inescapablenovelty screen induces a stress response and aversive stimuli ornegative affective states result in 22 kHz vocalizations. In addi-tion, we hypothesized the novelty place preference and 50 kHzvocalizations would be positively correlated, because animals thatengage the novel context find the novel context more rewardingwill vocalize more at 50 kHz. Our results indicate that globally,the individual difference screens were not correlated, suggestingthey were each measuring a different individual difference traitand that novel behavior in the rat is complex. However, harmonicand frequency modulated (FM) changed differently with repeatedtickling and demonstrate that subtypes of 50 kHz USVs can beused to understand attribution of incentive value associated withreward. Significant relationships were revealed between the nov-elty place preference screen and the change in harmonic 50 kHzUSVs measured during different phases of the experiment. Togetherthese results indicate that subtypes of 50 kHz USVs may be capa-ble of elucidating affective/motivational differences observed inthe novelty place preference screen and attribution of incentivevalue.

2. Methods

2.1. Animals

Fifty 30-day-old male, Sprague-Dawley rats were obtained fromCharles River Laboratories. All animals were housed in a temper-ature and humidity controlled colony room. Animals were housedindividually in transparent polyurethane cages with Carefreshrodent bedding with access to food and water ad libitum. Uponarrival all animals were handled, but not tickled, for ∼1 min daily tofacilitate experiment handling procedures. Experimentation began10 days after arrival. The colony room was maintained on a 12:12light–dark cycle, with lights on from 7:00 to 19:00. All behavioraltesting occurred in the light cycle. Based on previous literature, ani-

quency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

mals were tested in the light cycle [17,36,41]. All experimental pro-cedures were approved by the Kansas State University InstitutionalAnimal Care and Use Committee and complied with the Guide forthe Care and Use of Animals (National Research Council, 2011).

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.2. Apparatus

.2.1. Inescapable novelty screenLocomotor activity chambers measuring 46 × 46 × 46 cm were

sed to measure the locomotor activity in response to a novelnvironment. Each activity chamber wall was constructed of trans-arent plexigass and the plastic floor was covered in pine shavingsedding. The activity chamber was surrounded by a photobeamensor system placed approximately 2.5 cm above the plastic floornd created a 16 × 16 photocell array. Each cell was spaced 2.54 cmpart (Coulbourn Instruments, TruScan 2.01), and automaticallyeasured the amount of horizontal movement in cm. A white noise

enerator (70 dB) was used to create background noise to reducehe influence of external sounds.

.2.2. Novelty place preference screenEach place preference chamber was partitioned into three

ompartments. The walls of the chamber were constructed of plex-glass. The end compartments of the apparatus were 29 × 23 × 45L × W × H) cm. One end of the compartment was black and theoor of this compartment was constructed of 15 metal rods (6 mm

n diameter), spaced 2 cm apart. Compressed pellet bedding wassed in the litter tray under this compartment. On the oppositend, the compartment was white and the floor was constructedf wire mesh (13 × 13 mm grids). Pine shavings bedding was con-ained in the litter tray under this compartment. Between the blacknd white end compartments was a smaller center compartment9 × 23 × 45 (L × W × H) cm. The walls and floor were painted graynd constructed of plexiglass. On the test day the walls that parti-ioned the apparatus into three compartments were replaced withartitions allowing access to all compartments with an opening of0.5 × 10.5 cm.

.2.3. Ultrasonic arena and technical equipmentAll ultrasonic recording were completed in a separate room

ree of external noise contamination and of vocalizations producedy other animals. The heterospecific screen was completed in atandard transparent shoebox cage covered with Carefresh bed-ing. Ultrasonic vocalizations were recorded using an ultrasonicicrophone (Ultramic 200K, dodotronic.com) and SEAwave recor-

ing software. Analysis of each sound file was completed usingviSoft SASLab Pro Bioacoustics sound analysis software. Ultra-onic vocalizations of all call types were counted automaticallynd scored similar to Ahrens et al. [25] and Burgdorf et al. [38].he Ultramic 200K and recording software were interfaced with

separate computer that automatically saved each sound file forater analysis. Briefly, each sound file was opened in SASLab Prond the software generated a spectrogram. AviSoft software auto-atically marked and scored the parameters of each potential USV

f all call types ranging from 18 to 100 kHz and having a dura-ion of at least 10 ms. A trained researcher identified the shape ofhe USVs and scored them as flat, harmonic or frequency modu-ated (FM). During shape identification all USVs were verified usinghe playback function in the SASLab Pro software. Flat USVs wereperationally defined as having no visual fluctuations. HarmonicSVs were operationally defined as having 1 or 2 fluctuations,hange in frequency, and/or a complex shape that was not flatr FM, including step-like USVs. Frequency modulated USVs wereperationally defined as having 3 or more fluctuations and includedrill USVs. The USV parameter values (duration, start/end time,mplitude, frequency) were copied to Microsoft Excel and USVs

Please cite this article in press as: Garcia EJ, et al. Harmonic and freqconditioned and unconditioned reward processing. Behav Brain Res (2

ere categorized into 22 kHz (20–28 kHz) or 50 kHz (35–100 kHz)ased on peak frequency. Calls that had a peak frequency in theange of 28,001–34,999 Hz were not classified as 22 or 50 kHz USV.he actual number of USVs left unclassified was small, ∼3–5%,

PRESSResearch xxx (2015) xxx–xxx 3

depending on the animal. Over 25,300 USVs were scored in thepresent study.

2.3. Experiment 1: NSS and 50 kHz USVs relationship

2.3.1. Individual difference screens procedureTwenty animals were screened in the order of: (1) inescapable

novelty screen, (2) novelty place preference screen and (3) het-erospecific screen. The order of the screens was determined byprevious literature and in order of least to most invasive.

2.3.2. Inescapable novelty screenAnimals were transported into a separate testing room housing

locomotor activity chambers. Animals were subsequently placedinside the novel environment and locomotor activity was measured(total distance traveled cm) automatically for a duration of 30 min.

2.3.3. Novelty place preference screenThe following day animals were transported into a separate test-

ing room and were screened with the novelty place preference.During habituation animals were placed in either the white or blackside of the place preference chamber for 30 min for two consecutivedays. On the third day, the partition restricting access to the habitu-ation side was removed, and animals had the choice to explore thenovel environment for 15 min. Animals were placed into the neutralgray area upon being placed inside of the place preference cham-ber. Time spent in the habituated and novel environments wasrecorded and a preference ratio was calculated for each animal. Theratio was calculated: (time spent in novel)/(time in novel + time inhabituated). In addition to the preference ratio calculation, latencyto enter habituated side, and latency to enter the novel side weremeasured. Entry into a compartment was operationally defined asboth front paws in the same compartment.

2.3.4. Heterospecific USV screenAnimals were transported into a holding room adjacent to the

ultrasonic recording room. Animals were then individually broughtinto the ultrasonic recording room, which was free of other animalsand external noise that would impede ultrasonic recording. Theheterospecific USV screen was conducted by mimicking conspecificjuvenile play. The experiments ‘wrestled’ and ‘tickled’ the animalwith fast-finger movements alone the nape of the neck, dorsal andventral sides of the animal for 15 s, followed by 15 s of no stimula-tion. The stimulation cycle was conducted for a total of 2 min. Eachanimal experienced 3 days of ‘tickling’ to habituate the animal tothe tickle procedure. On the 4th day an identical procedure wasused with the exception that the ultrasonic microphone (Ultramic200K) was ON and recorded USVs of all call types.

2.3.5. Experiment 1 analysesPearson r correlational analyses were used to determine the

relationships between the behavioral responses for each of the indi-vidual difference screens. Additionally, correlational analyses wereused to determine the relationships between the types of USVsand the inescapable novelty screen and the novelty place prefer-ence screen. For computation, the total counts for each USV typewere correlated. For all correlational analyses significance was setat p < .05. Flat USVs and 22 kHz USV were generally not observed.Therefore analyses for these USVs could not be completed and theywill not be discussed in subsequent analyses.

2.4. Experiment 2: NSS relationship with repeated USV recordings

uency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

2.4.1. Individual difference screen procedureThirty naïve rats were screened in Experiment 2. The individ-

ual difference screens procedure was identical to Experiment 1,

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Table 2Experiment 1: Correlations between the inescapable novelty and novelty place pref-erence screens with 50 kHz USV divided into harmonic and frequency modulatedcall types.

Measure IEN NPP Harmonic FM

IEN – .05 −.11 .15NPP – −.05 −.15

screen (r(28) = .40, p = .03). Further examination of this significantrelationship indicates it was due to the change in harmonic USVs(r(28) = .37, p = .05), and not a change in FM USVs (r(28) = .27, p = .15;see Table 4).

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xcept for additional USVs recordings. Additional recording weresed to fully elucidate the relationship between USVs and NSS, ando examine if these relationships changed as a function of exposureo a novel versus conditioned chamber or as a function of acuteersus repeated tickle stimulation. Ultrasonic vocalizations of allall types were recorded four separate times for 2 min during theeterospecific screen. The first recording was completed upon thenimal’s first entry into the tickle arena and they were not tickled,hese are termed Novel USVs (N-USV). The second recording wasompleted during the animal’s first exposure to tickling, these areermed Acute USVs (A-USV). After 3 days of tickling habituation,he third and fourth recordings were completed. The third recor-ing was completed upon entry into the tickle arena without anyickling, these are termed Conditioned USVs (C-USV). The fourthecording was completed during the animal’s fourth exposure toickling and these are called Repeated USVs (R-USV).

.4.2. Experiment 2 analysesFor experiment 2, Pearson r correlational analyses were used

o determine the relationships of USV types measured at differentime points and the novelty screens. The total counts of each typef USV were used in the correlation analyses. In addition, repeatedeasures 1-way ANOVA was used to determine if total 50 kHz USVs

hanged during recording sessions. Significant differences revealedy the omnibus ANOVA were probed with post hoc Bonferroniomparisons. Bonferroni comparisons controls for Type I error byividing alpha by number of comparisons (p < .05/6 = .008). Similar-way ANOVA and post hoc tests were used to examine if harmonicr FM changed across recording sessions. It should be noted that flatSVs and 22 kHz USV were generally not observed. Therefore anal-ses for these USVs could not be completed and they will not beiscussed in subsequent analyses.

. Results

.1. Experiment 1: NSS and 50 kHz USVs relationship

.1.1. Relationship between individual difference screensThe inescapable novelty screen and 22 kHz vocalizations were

ot related as evidenced by the low frequency of 22 kHz USVsdata not shown). The inescapable novelty screen and heterospe-ific screen did not show any relationship (r(18) = .07, p = .77). Theeterospecific screen and the novelty place preference were alsoot correlated (r(18) = −.10, p = .70). Previous research has showno relationship between the responses in the inescapable noveltycreen and the novelty place preference, and that finding was repli-ated (r(18) = .06, p = .81; see Table 1).

.1.2. Relationship between the screens and USV subtypesFurther examination of the relationship between the sub-

ypes of USVs (FM and Harmonic) and the inescapable noveltycreen revealed no significant relationships (r(18) = .15, p = .54;

Please cite this article in press as: Garcia EJ, et al. Harmonic and freconditioned and unconditioned reward processing. Behav Brain Res (2

(18) = −.11, p = .63) respectively. Similarly, the relationshipetween the number of FM USVs and harmonic USVs with the nov-lty place preference response revealed no significant relationshipsr(18) = −.15, p = .54; r(18) = .05, p = .85), respectively. Frequency

able 1xperiment 1: Correlations between the individual differences screens. IEN:nescapable novelty, NPP: novelty place preference, heterospecific: 50 kHz USV inesponse to heterospecific screen.

Measure IEN NPP Heterospecific

IEN – .06 .07NPP – −.10Heterospecific –

Harmonic – .42FM –

modulated and harmonic USVs did not show a significant relation-ship, but was trending toward a positive relationship (r(18) = .42,p = .07; see Table 2). These results suggest that the inescapablenovelty screen, novelty place preference screen, and heterospecificscreen are measuring different aspects of novelty as evidenced bytheir lack of relationship.

3.2. Experiment 2: NSS with repeated USV recordings

3.2.1. Relationship between individual difference screensThe inescapable novelty screen and the novelty place preference

screen were not correlated (r(28) = .03, p = .87). The inescapablenovelty screen and novelty place preference screen were not cor-related with 50 kHz USVs recorded at any time (e.g. N-USV, C-USV,A-USV and R-USV, see Table 3 for summary correlations). However,there were significant correlations observed between 50 kHz USVsin each of the recording sessions. N-USV showed significant correla-tions with A-USV, C-USV, and R-USV (r(28) = .72, p < .001; r(28) = .58,p = .001; r(28) = .59, p = .001), respectively. A-USV showed a sig-nificant correlation with C-USV and R-USV (r(28) = .64, p < .001;r(28) = .85, p < .001). Finally, C-USV showed a significant correlationwith R-USV (r(28) = .75, p < .001).

3.2.2. Relationship between the screens and USV subtypesTo fully determine the relationship between novelty and USV,

the subtypes of the USV were correlated with the novelty individualdifference screens across each of the four USV recording sessions.The inescapable novelty response and the novelty place prefer-ence were not correlated with FM or Harmonic USVs recordedduring the N-USV, A-USV, C-USV, or R-USV sessions. A differ-ence score for FM and harmonic was calculated to examine if thechange from non-tickled environment (�E = C-USV − N-USV) or thetickling (�T = R-USV − A-USV) was correlated with either noveltyscreen. The correlational analyses indicated that �T for harmonicand FM was not correlated with the inescapable novelty screenand the novelty place preference screen, all p’s > .05. Alternatively,the �E was positively correlated with the novelty place preference

quency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

Table 3Experiment 2: Correlations between the individual difference screens. IEN:inescapable novelty, NPP: novelty place preference, N-50: 50 kHz USV in responseto novel environment, A-USV: 50 kHz USV in response to first day of tickling, C-50:50 kHz USV in response to tickle-associated context, R-USV: 50 kHz USV in responseto repeated tickling.

Measure IEN NPP N-USV A-USV C-USV R-USV

IEN – .03 −.03 −.19 −.18 −.17NPP – −.08 .15 .27 .08N-USV – .72** .58** .59**

A-USV – .64** .85**

C-USV – .75**

R-USV –

** p < .001.

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Table 4Experiment 2: Correlations between individual difference screens: IEN: inescapablenovelty, NPP: novelty place preference, FM: frequency modulated 50 kHz USV, Har-monic: harmonic 50 kHz USV, �E refers to the difference: C-USV − N-USV, �T refersto the difference: R-USV − A-USV.

Measure IEN NPP �E-FMUSV �E-HarmUSV �T-FMUSV �T-HarmUSV

IEN – .03 −.03 −.21 .13 .02NPP – .27 .37* .06 −.22�E-FMUSV – .33 .06 .26�E-HarmUSV – .47** .14�T-FMUSV – .03�T-HarmUSV –

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Fig. 1. Experiment 2: The total number of 50 kHz USV across the four recordingsessions for the H-USV screen. Different letters above (A, B, and C) are significantlydifferent from each other letter using Bonferroni post hoc comparisons, p < .008.Columns with same letter are not different.

Fig. 2. Experiment 2: Only FM 50 kHz USV across the four recording sessions for theH-USV screen. Different letters above (A, B, and C) are significantly different from

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* p < .05.** p < .01.

Upon further examination of the FM and Harmonic USV sub-ypes, it became clear that FM USVs were highly correlated acrosshe recording sessions. Similarly, harmonic USVs were stable afterhe first tickling trial (A-USV) and remained highly correlated acrosshe recording sessions, but FM and Harmonic USV were not cor-elated with each other across any of the recording sessions (seeable 5). Taken together, these results suggest that high USV call-ng animals are not simply calling at high rates randomly, instead ituggests that USV calling is purposeful and remained stable acrossecording sessions.

.2.3. Total 50 kHz USV across recording sessionsThe 1-way repeated measures ANOVA revealed that there was at

east one difference in total 50 kHz USV between the recording ses-ions (F(3, 87) = 52.81, p < .001). Bonferroni post-hoc comparisonsndicated that there was a significant difference between N-USVnd C-USV, p < .001, with more 50 kHz USV during the C-USV recor-ing. Animals vocalized more during the A-USV than the N-USVecording session, p < .001. The A-USV was not different from the C-SV screen, p = .38. Repeated tickling (R-USV) induced more 50 USV

han the A-USV, C-USV, and N-USV recording sessions, all p’s < .001.o summarize, the animals vocalized more upon entry into a con-itioned rewarding environment, but animals showed the greatestumber of 50 kHz USV is response to repeated tickling (R-USV; seeig. 1).

.2.4. FM 50 kHz USV across recording sessionsTo examine how FM 50 kHz USV were changing across recording

essions, a 1-way repeated measures ANOVA was used to comparehe means of FM 50 kHz USV across each of the recording sessions.he analysis revealed at least one significant difference in mean

Please cite this article in press as: Garcia EJ, et al. Harmonic and freqconditioned and unconditioned reward processing. Behav Brain Res (2

M USV between the recording sessions (F(3, 87) = 41.86, p < .001).onferroni post hoc comparisons indicated that FM USVs were notifferent during the N-USV and C-USV recording sessions. However,M USVs recorded during the N-USV were significantly less than the

able 5xperiment 2: Correlations between individual difference screens: IEN: inescapable noveltarmonic 50 kHz USV in each recording condition, novel, acute, conditioned, and repeate

Measure IEN NPP FM N-USV Harm N-USV FM A-USV

IEN – .03 −.19 .03 −.10

NPP – −.34 .02 −.20

FM N-USV – −.04 .68**

Harm N-USV – .12

FM A-USV –

Harm A-USV

FM C-USV

Harm C-USV

FM R-USV

Harm R-USV

* p < .05.** p < .01.

427

each other letter using Bonferroni post hoc comparisons, p < .008. Columns withsame letter are not different.

A-USV and R-USV recording sessions, all p’s < .001. Similarly, therewas a significant increase in FM USVs from the C-USV to the A-USVand R-USV recording sessions, p < .001. Taken together these resultsindicate that acute and repeated tickling induces more FM USVsthan a novel or tickle-conditioned context. Finally, repeated tick-ling (R-USV) significantly increased in FM USVs when compared tothe A-USV, p < .001 (Fig. 2). These results provide evidence that FM

uency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

50 kHz do not change in response to Pavlovian conditioned context.However, FM 50 kHz USVs increased across the other recording ses-sions, suggesting that they are sensitive to the tickle stimulation

y, NPP: novelty place preference, FM: frequency modulated 50 kHz USV, Harmonic:d.

Harm A-USV FM C-USV Harm C-USV FM R-USV Harm R-USV

−.17 −.18 −.13 .00 −.22.24 −.02 .29 −.10 .19

−.03 .48* −.04 .64** −.06.72** −.28 .68** .04 .65**

.15 .56** .12 .83** .08– −.31 .73** .14 .89**

– −.01 .55* −.28– .25 .81**

– .13–

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Fig. 3. Experiment 2: Only harmonic 50 kHz USV across the four recording sessionsffw

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or the H-USV screen. Different letters above (A and B) are significantly differentrom each other letter using Bonferroni post hoc comparisons, p < .008. Columnsith same letter are not different.

tself, but are not induced by the conditioned context associatedith the tickling procedure.

.2.5. Harmonic 50 kHz USV across recording sessionsA 1-way repeated measures ANOVA was used to compare the

ean harmonic USVs during the recording sessions, and the analy-is revealed there was at least one difference in responding (F(3,7) = 36.02, p < .001). Post hoc Bonferroni comparisons revealedhat harmonic 50 kHz USVs recorded during the N-USV were signif-cantly less than the C-USV, A-USV and R-USV recording sessions,ll p’s < .001. Harmonic USVs did not change across any of the otherSV recording sessions, all p’s > .10. Importantly, harmonic USVs

ncreased from N-USV to C-USV suggesting that harmonic USVshanged in response to a Pavlovian conditioned context. Theseesults indicate that acute tickling induced more harmonic 50 kHzSV when compared to the novel non-tickled recording sessions,ut harmonic 50 kHz USV do not increase with repeated tickling tri-ls and are specifically induced by a Pavlovian conditioned contextsee Fig. 3).

. Discussion

The present study aimed to understand the relationshipetween novelty and sensation seeking (NSS) and USVs. Ouresearch replicated previous work that the inescapable noveltycreen and novelty place preference are not correlated, indicatinghey are likely measuring different aspects of NSS or are mea-uring different individual difference traits [14,15,17]. Globally,otal 50 kHz USVs were not correlated with either the inescapableovelty screen or novelty place preference. However, deeper exam-

nation revealed the change in harmonic USVs while not beingickled (N-USV to C-USV) are correlated to the novelty place pref-rence screen, and not the inescapable novelty screen. This results in corroboration with Taracha [29], that determined USVs areot related to the inescapable novelty screen, however our dataemonstrate a significant relationship with novelty place pref-rence screen. Interestingly, this significant correlation was onlyresent when the animals were not tickled. Finally, FM and har-onic USVs were not correlated, suggesting they are unique and

ould be used to understand different affective or motivationaltates.

Novelty and sensation seeking in the rat has been modeled using

Please cite this article in press as: Garcia EJ, et al. Harmonic and freconditioned and unconditioned reward processing. Behav Brain Res (2

he inescapable novelty screen, novelty place preference screen,nd novel object recognition task (or spontaneous object recogni-ion [42]). Although all of these measures are thought to measureovelty behavior in the rat, the inescapable novelty screen, novelty

PRESSResearch xxx (2015) xxx–xxx

place preference, and novel object recognition task are often foundto not be correlated likely suggesting that the novelty screens aremeasuring different aspects of novelty or measuring different indi-vidual difference traits. It should be mentioned that other work byCain [7] and Beckman [43] has shown that the inescapable nov-elty screen and novelty place preference are negatively correlated.Whether the inescapable novelty screen and novelty place prefer-ence screen are negatively correlated or not correlated does lendsupport that the screens are measuring different traits, yet it is notdetermined if those are encompassed in novelty per se or whetherthey are indicative of two entirely different traits.

The inescapable novelty screen has been shown to increase cor-ticosterone which lends support that the stress axis is activatedduring this novelty screen. Interestingly, the elevation in cortico-sterone is positively correlated with locomotor activity, and thoserats classified as HR have an increase in corticosterone that persistsfor up to 2 h when compared to LR [13]. Therefore, it is possible thatthe inescapable novelty screen is more reflective of a response tostressful environment and possibly a dysfunctional HPA axis ratherthan response to a novel environment.

Unlike the inescapable novelty screen, the novelty place pref-erence screen has not been shown to induce a stress response.In this screen the animals have the choice to engage a novelenvironment. Dopamine activity specifically in the nucleus accum-bens shell shows an influx when an animal chooses to engage anovel environment over a familiar environment. Importantly, thedopamine influx was only transiently increased upon entry into thenovel environment the first time [16]. Other microdialysis evidencefrom Legault and Wise [44] suggests that the increase in dopaminepersists for 2 h. The differences in how long dopamine levels are ele-vated could be due to the habituation period, because Rebec [16]habituated animals for 30 min for 3 days, whereas Legault and Wise[44] habituated the animals for 18 h. Therefore, the difference indopamine influx may be due to the degree of habituation or theinverse, how novel the stimulus is, providing strong support thatnovelty in the rodent is complex.

Novelty and sensation seeking behavior activates dopaminetransmission in the mesolimbic dopamine pathway. For this reason,NSS behavior is thought to be rewarding to the animal, howeverit appears that NSS can be dichotomized into at least two behav-ioral outcomes. The locomotor effects of novelty, which seem to bemediated by D2 dopamine receptors and activation of HPA axis; andsecond, namely the incentive effects of novelty which are mediatedby D1 dopamine receptors [5,45]. Although it should be mentionedthat the reinforcing and locomotor effects of novelty or psychomo-tor stimulants is not completely dissociable [14]. With this in mind,it is possible that the inescapable novelty screen and the noveltyplace preference screen are measuring the locomotor effects andincentive effects of NSS behavior, respectively, which may explaintheir lack of relationship with each other. An alternative expla-nation is that the inescapable novelty screen may be more repre-sentative of an escape behavior [14]. Therefore, the two individualdifference screens are independent and account for different indi-vidual differences [14,15,17]. The inescapable novelty and noveltyplace preference screens may be measuring a propensity for ‘druguse prone’ phenotype and ‘drug addiction prone’ phenotype [15].

Previous research and the results of the present study indicatethat NSS involve more than one aspect, therefore it could be benefi-cial to understand how positive and negative affective processes arerelated to NSS behavior. Twenty-two kHz USVs occurred at a verylow call rate or were not observed. Examination of the data prior toany statistical tests determined many animals did not make these

quency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

calls and therefore the correlation could not be reliably computed.This indicates that 22 kHz USVs were, for the most part, not evokedby a novel environment, acute tickling, an environment associatedwith tickling, or repeated tickling. This result provides evidence

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hat at no time during the heterospecific screen (N-USV, A-USV,-USV, or R-USV) animals were in an aversive affective or moti-ational state. In addition, the result suggests that 22 kHz USVs areot related to either the inescapable novelty screen or novelty placereference screen. Although, the present study did not measureSVs during the inescapable novelty screen, however, entry into

he tickle apparatus was novel and unavoidable, which is sufficiento elevate corticosterone and activate the stress axis. It was thoughthat 22 kHz USVs observed during the heterospecific screen wouldhow a positive relationship with the inescapable novelty screenut that relationship was not found. An alternative explanation forhe lack of relationship could be that the environment did induce atress response, but was not great enough to surpass the thresholdeeded to evoke 22 kHz USV. Even cocaine withdrawal is insuffi-ient to increase 22 kHz USV alone and requires administration oftartling mild air puffs [46]. Therefore, measuring the emission of2 kHz USVs may not be adequate to uncover differences in affec-ive state between higher and lower responders in the inescapableovelty screen.

The present study also determined that 50 kHz USVs increasever repeated tickling administrations, and supports previousesearch by Burgdorf [36]. However, we note the increase wasriven by an increase in FM USVs. This result suggests thatepeated tickling induces FM USVs and is indicative of an increasedehavioral response to a natural reinforcer. The increase inM vocalizations is similar to results seen with intravenousmphetamine exposure [25], and suggests that tickling can induceehavioral sensitization, as evidenced by the significant increase inM USV from the A-USV to the R-USV recording sessions. However,anipulations to determine if these behavioral effects are long-

asting were not determined in this study. Traditionally, behavioralensitization is examined through non-contingent drug adminis-ration and locomotor response assessment. Typically, previousxposure to drugs of abuse results in an increase in locomotoresponse and this elevated locomotor response occurs throughout

variety of stimulant drugs. Furthermore, the neurological changesnderstood to take place after administration of drugs of abusere thought to persist for long durations of time, and can becomectivated by contextual cue and motivate drug seeking behavior.onetheless, evidence from other research [29,37,47] indicates that0 kHz USV and locomotor activity are distinct behaviors, and indi-ates that behavioral sensitization can occur in different domains,ossibly motor and affective. Other research indicates systemicmphetamine injection locomotor response is moderated by USVall rate [48] and repeated intravenous amphetamine increasesearing behaviors that coincide with FM USV sensitization, albeitM USV sensitization occurred before rearing sensitization [25].aken together, it is likely that FM USV sensitization occurs outsidef locomotor or other motor sensitization behaviors, and FM sen-itization can occur in response to repeated non-pharmacologicalanipulations such as tickling.Unlike the change observed in FM USVs, harmonic USV were not

hanged by repeated tickling. Instead, our data suggest these USVsre induced upon receipt of acute reward, but quickly are asso-iated with contextual stimuli. After the acute tickling exposure,armonic vocalizations remained elevated upon re-entry into theickle arena, demonstrating a Pavlovian association. This was par-icularly interesting because entry into a novel environment didot induce as many harmonic USVs, and animals were not tickleduring the C-USV recording session. Yet, the difference between the-USV and C-USV recording sessions was positively correlated to

he novelty place preference. Thus, it is apparent that the tickle

Please cite this article in press as: Garcia EJ, et al. Harmonic and freqconditioned and unconditioned reward processing. Behav Brain Res (2

rena acquired incentive value, as evidenced by the increase inositive affective harmonic USVs. Most importantly, it was the har-onic USVs and not the FM USVs that were positively correlated

o the novelty place preference screen. Interestingly, animals that

PRESSResearch xxx (2015) xxx–xxx 7

attribute incentive value to an environment as measured by thenovelty place preference screen are also more likely to attributeincentive value to a food cue and to be classified as sign track-ers in a Pavlovian task. These rats are also more sensitive to lowunit doses of cocaine (Beckman). Additional research suggests theremay be a relationship between incentive value and USVs. Ani-mals that emit 50 kHz USVs show a stronger conditioned placepreferences amphetamine, cocaine and DAMGO an opioid agonist[37–39]. These results suggest that 50 kHz USVs can be used todetermine the magnitude of incentive value attributed to the con-text and USVs predict subsequent conditioned place preferencebehavior.

Harmonic USVs were observed in all recording sessions of theheterospecific screen, but they were preferentially induced by thetickle associated environment, and the change in harmonic USVswas positively correlated to the novelty place preference screen.Novelty and sensation seeking can be represented by at least twobehavior domains: the locomotor effects and the incentive effects,with each mediated by D2 and D1 dopamine receptors respectively.The novelty place preference screen has been shown to predict thetransition to compulsive drug taking, while the inescapable nov-elty screen has not shown a similar relationship, suggesting thatthe incentive value associated with drug reward is important forcompulsive drug taking [15]. Interestingly, 50 kHz USVs are evokedby a conditioned stimulus associated with a palatable food. Theseanticipatory 50 kHz USVs were reduced dose dependently by SCH23390 a D1 antagonist and opioid antagonism using naltrexone.Taken together, this evidence suggests that the incentive value ofthe tickle associated environment as measured by harmonic USVsin the current study are mediated by D1 dopamine receptors. Col-lectively, harmonic USVs could be used in conjunction with thenovelty place preference screen to understand attribution of incen-tive value to other conditioned stimuli in future studies. Given thatharmonic and FM USVs differentially changed during the recor-ding sessions and were generally not correlated, it is likely they aremeasuring different affective motivations. One possibility is thatharmonic and FM USVs represent ‘wanting’ and ‘liking’, respec-tively. While 50 kHz USVs have been associated with a positiveaffective state, examination of the call subtypes have not beenfully determined. Therefore, it is necessary to determine if specificsubtypes of USVs are correlated with other behavioral measuresto evaluate the relationship between the call subtypes and affec-tive and motivational states. Evaluation of the subtypes of USVswill provide real-time measurements of affective and motivationalstate that can be used to quantify individual differences and predictmaladaptive behaviors.

5. Conclusion

The present study confirmed that NSS is a complex behavior.The novelty place preference screen, a screen known to predict thetransition to compulsive drug taking, was correlated with harmonic50 kHz USVs, while FM USVs were specifically evoked by the tick-ling. These results indicate that harmonic USVs are responsive to anenvironment associated with reward and that FM USVs are respon-sive to the receipt of such a reward. Therefore, our data indicate thatUSVs of multiple call types should be examined, because they revealdifferences in how reward is anticipated, attributed to contextualstimuli, or experienced.

uency modulated ultrasonic vocalizations reveal differences in015), http://dx.doi.org/10.1016/j.bbr.2015.03.049

Acknowledgements

The authors would like to thank Kathryn C. Johns and RichardTurner for their contributions to the experimental procedures.

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http://dx.doi.org/10.1016/j.neuropharm.2014.04.008.[48] Brudzynski SM, Gibson B, Silkstone M, Burgdorf J, Kroes RA, Moskal JR, et al.

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