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Hearing Research 332 (2016) 121e136

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Hearing Research

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Research paper

Modulation of auditory brainstem responses by serotonin and specificserotonin receptors

Melissa A. Papesh a, *, Laura M. Hurley b

a Indiana University, Department of Speech and Hearing Sciences, 200 South Jordan Avenue, Bloomington, IN 47405, USAb Indiana University, Department of Biology, Center for the Integrative Study of Animal Behavior, 1001 E. Third Street, Bloomington, IN 47405, USA

a r t i c l e i n f o

Article history:Received 23 May 2015Received in revised form28 October 2015Accepted 23 November 2015Available online 12 December 2015

Keywords:ElectrophysiologyEvent related potentialHearingMouseSerotonergicTone

Abbreviations: 5-HT, 5-hydoxytryptamine, serotoDPAT hydrobromide, 5-HT1A receptor agonist; ABR, aAEP, auditory evoked potential; ANOVA, analysis ofdecibel referenced to sensation level; dB SPL, decibellevel; IC, inferior colliculus; IP, intraperitoneal; SEM,CP93129, CP93129 dihydrochloride, 5-HT1B receptorchloride, 5-HT2A/C receptor agonist; Ketanserin, ketaceptor antagonist; NAS-181, (2R)-2-[[[3-(4-Morpholin8-yl] oxy]methyl] morpholinedimethanesulfonate, 5pCPA, 4-chloro-DL-phenylalanine methyl ester hydronin depletion agent; S-WAY, (S)-WAY 100135 dihydantagonist* Corresponding author. Present Address: Nation

Auditory Research Portland, VA Medical Center, 371Road, Portland, OR 97207, USA.

E-mail address: [email protected] (M.A. Pape

http://dx.doi.org/10.1016/j.heares.2015.11.0140378-5955/Published by Elsevier B.V.

a b s t r a c t

The neuromodulator serotonin is found throughout the auditory system from the cochlea to the cortex.Although effects of serotonin have been reported at the level of single neurons in many brainstem nuclei,how these effects correspond to more integrated measures of auditory processing has not been well-explored. In the present study, we aimed to characterize the effects of serotonin on far-field auditorybrainstem responses (ABR) across a wide range of stimulus frequencies and intensities. Using a mousemodel, we investigated the consequences of systemic serotonin depletion, as well as the selectivestimulation and suppression of the 5-HT1 and 5-HT2 receptors, on ABR latency and amplitude. Stimuliincluded tone pips spanning four octaves presented over a forty dB range. Depletion of serotonin reducedthe ABR latencies in Wave II and later waves, suggesting that serotonergic effects occur as early as thecochlear nucleus. Further, agonists and antagonists of specific serotonergic receptors had differentprofiles of effects on ABR latencies and amplitudes across waves and frequencies, suggestive of distincteffects of these agents on auditory processing. Finally, most serotonergic effects were more pronouncedat lower ABR frequencies, suggesting larger or more directional modulation of low-frequency processing.This is the first study to describe the effects of serotonin on ABR responses across a wide range ofstimulus frequencies and amplitudes, and it presents an important step in understanding how seroto-nergic modulation of auditory brainstem processing may contribute to modulation of auditoryperception.

Published by Elsevier B.V.

1. Introduction

Evidence accumulated over the past twenty years indicates thatserotonin plays an important role inmodulating auditory responses

nin; 8-OH-DPAT, 8-hydroxy-uditory brainstem response;variance; dB, decibel; dB SL,referenced to sound pressurestandard error of the mean;agonist; DOI, (±)-DOI hydro-nserin tartrate, 5-HT2A/C re-ylmethyl)-2H-1-benzopyran--HT1B receptor antagonist;chloride, endogenous seroto-rochloride, 5-HT1A receptor

al Center for Rehabilitative0 SW, US Veterans Hospital

sh).

with potentially significant functional consequences. Serotonin hasbeen linked to clinical auditory disorders such as tinnitus (Nore~naet al., 1999; Simpson and Davies, 2000; Caperton and Thompson,2011) and hyperacusis (Marriage and Barnes, 1995; Attri andNagarkar, 2010), as well as the auditory manifestations associatedwith non-auditory-specific disorders including migraine (Goadsby,1998; Hamel, 2007; Panconesi, 2008; Sand et al., 2008), depression(Gopal et al., 2000; Chen et al., 2002; Kampf-Sherf et al., 2004;Gopal et al., 2005), schizophrenia (Bleich et al., 1988; Breier, 1995;Park et al., 2010), and post-traumatic stress disorder (Southwicket al., 1999; van der Kolk, 2001; Lee et al., 2005), among others.Associations between serotonergic activity and auditory respon-sivity have been supported in part by auditory evoked potential(AEP) measures assessing cortical function. Here, changes inendogenous serotonin levels have been linked to changes in peakcomponent amplitudes (Ehlers et al., 1991; Manjarrez et al., 2005)and latencies (Concu et al., 1978) as well as dynamic variation inresponses to stimuli across time (Johnson et al., 1998; Stevens et al.,2006) and stimulus level (Hegerl and Juckel, 1993; Juckel et al.,

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M.A. Papesh, L.M. Hurley / Hearing Research 332 (2016) 121e136122

1997; Juckel et al., 1999; Gallinat et al., 2000; Bruder et al., 2001;Hegerl et al., 2001; Jaworska et al., 2013). These studies generallysupport the theory that reduced serotonergic function is associatedwith increased activity and responsivity in the auditory cortex.Effects of serotonin lower within the auditory pathway have alsobeen demonstrated in numerous extra- and intracellular studies(Ebert and Ostwald, 1992; Mizutani et al., 2006; Wang andRobertson, 1997; Fitzgerald and Sanes, 1999; Faingold, 1999;Hurley and Pollak, 1999; Hall and Hurley, 2007; Miko and Sanes,2009), indicating the likelihood that serotonergic effects beginwell before the level of the cortex. Auditory brainstem responses(ABRs) can serve as a versatile tool allowing comparison of sero-tonergic effects across levels of analysis. Since ABRs represent thesummed activity across large populations of auditory neurons, theycan illustrate whether effects observed at the level of single neu-rons are prominent enough to influence the amplitude or syn-chrony of population activity. ABRs can also be used to compare theinfluence of serotonergic manipulation at different sites along theauditory neuraxis, a type of information often used to identifypotential sites of origin for auditory phenomena (Starr and Achor,1975; Stockard and Rossiter, 1977). Finally, ABRs can be comparedbetween human subjects and animal models of specific auditorydisorders, facilitating testing of hypotheses on the mechanistic roleof serotonin in disorders like tinnitus or hyperacusis (Tziridis et al.,2015; Heeringa and van Dijk, 2014). Despite these advantages, theeffects of serotonin, and particularly specific receptor pathways, onABR remain largely unexplored.

Serotonergic fibers and varicosities are abundant in the cochleaand each level of the auditory brainstem (Steinbusch, 1981; Willardet al., 1984; Fitzpatrick et al., 1989; Klepper and Herbert, 1991;Harvey et al., 1993; Gil-Loyzaga et al., 1997; Hurley and Thomp-son, 2001; Thompson and Hurley, 2004; Papesh and Hurley, 2012).Though fewer in number, some physiological investigations ofserotonergic effects in the brainstem support a suppressive orpartially suppressive role such that application of serotonin leads toreduced spontaneous and driven firing in the cochlear nucleus(Ebert and Ostwald, 1992), the trapezoid body (Mizutani et al.,2006), the superior olivary complex (Fitzgerald and Sanes, 1999),and the inferior colliculus (IC) (Hurley and Pollak, 2001; -Wanget al., 2008). In the IC, serotonin affects first-spike latencies andinterspike intervals (Hurley and Pollak, 2005; Hurley, 2006) as wellas changes in frequency tuning most often resulting in reducedresponse areas following serotonin appliction (Hurley and Pollak,2001; 2005; Hurley, 2006; Hall and Hurley, 2007). ABRs provide auseful tool by which to study the aggregate effects of serotonergicmanipulations across a large population of cells from the auditorynerve to the IC. Thus, a main goal of the present study was to useABRs to characterize the effects of endogenous serotonin depletionin the mouse model across a wide range of stimulus frequenciesand intensities, with the prediction that low levels of endogenousserotonin should elicit larger response amplitudes and shorterresponse latencies compared to baseline measures. Since the den-sities of serotonergic fibers are reported to be greater in low-frequency regions of several brainstem nuclei (Klepper andHerbert, 1991; Hurley and Thompson, 2001; Hurley et al., 2002;Papesh and Hurley, 2012), a further prediction is that depletion ofserotonin should have larger effects on low-frequency than onhigh-frequency responses.

Although the effects of serotonin in the auditory brainstem areoften suppressive, multiple studies reveal subsets of cells showingthe opposite trend of increased responsivity with serotonin appli-cation. The diversity in serotonergic effects is largely mediated bythe type and distribution of serotonergic receptors present on pre-and post-synaptic cells. Of the seven serotonin receptor families,the 5-HT1 and 2 families have been well-documented throughout

the auditory system, including the periphery. Predominant effectsof 5-HT1A and 5-HT2A/C receptor stimulation in the IC includereduced spiking and increased firing latencies in most cells testedin the central nucleus of the IC (Hurley, 2006, 2007), and increasedfrequency and amplitude of GABAergic postsynaptic currents(Wang et al., 2008). In contrast, stimulation of the 5-HT1B receptoris reported tomost often elicit excitatory effects such as increases inbandwidth of frequency response maps, higher firing rates, andlower thresholds of auditory neurons (Hurley, 2006; Hurley et al.,2008; Ramsey et al., 2010). Thus, a second goal of the presentwork is to begin an exploration of the role of multiple specificserotonergic receptors, the 5-HT1A, 1B, and 2A/C, on the modula-tion of ABRs. Although this is complicated by the fact that even thesame type of serotonin receptor may have different effects indifferent brainstem nuclei, a general prediction was that manipu-lating different types of serotonin receptor will create differentsuites of effects on the ABR.

2. Methods

2.1. Subjects

Subjects in the current study were male CBA/J (Jackson Labo-ratories) mice. This inbred strain is frequently chosen for auditorystudies due to its good hearing sensitivity maintained across thelifespan (Mikaelian and Ruben, 1965; Willott et al., 1991). A sche-matic of subject groups and experimental procedures is shown inFig. 1. A total of 56 subjects were tested at an average age of 2.3months (standard deviation of 0.6) at baseline testing. Twenty-fourof these subjects underwent depletion of endogenous serotonin.Following serotonin depletion, these 24 subjects were furtherdivided into three groups of eight, each of which received anagonist of the 5-HT1A, 1B, or 2A/C receptor. An additional 24 sub-jects were placed into three groups of eight with each groupreceiving an antagonist of either the 5-HT1A, 1B, or 2A/C receptor.Finally, the last eight subjects received saline injections but noserotonergic manipulations. This group served as a control toensure that changes recorded after serotonin depletion were notdue to the stress of injections or handling of animals. All protocolswere approved by the Bloomington Institutional Animal Care andUse Committee and were carried out in accordance with EUDirective 2010/63/EU.

2.2. Serotonergic pharmaceutical agents

Depletion of endogenous serotonin was achieved by intraperi-toneal injection of 4-Chloro-DL-phenylalanine methyl ester hy-drochloride (pCPA; Sigma Aldrich, Cat#C3635), which inhibits thefunction of tryptophan hydroxylase (Koe and Weissman, 1966).Repeated application of pCPA leads to progressive decreases inendogenous serotonin levels as serotonin stores are released andmetabolized, but not replenished (Dailly et al., 2006). Subjectsreceived a dose of 150 mg/kg pCPA diluted in physiological salineand administered intraperatoneally (i.p.) once every 24 h for sixdays, a regimen of pCPA injections that reduces endogenous sero-tonin levels by approximately 80e90% in mice (Dailly et al., 2006)while minimizing impact on nonserotonergic systems (Bauer et al.,2002). Because pCPA is reported to reduce appetite (Bubenik andPang, 1993), subjects’ weight was monitored daily to ensure thatany changes in appetite resulting from pCPA did not cause undueweight loss.

To explore the role of specific serotonergic receptors on ABRmeasures, selective agonists and antagonists of the 5-HT1A,1B, and2 receptors were administered to designated subject groups (Fig. 1).Agonists were administered to subjects only after serotonin

Fig. 1. Schematic of subject groups and timeline of experimental procedures. Subject groups are shown in black circles, ABR measurements are shown in white boxes, andpharmaceutical doses are shown in gray boxes.

M.A. Papesh, L.M. Hurley / Hearing Research 332 (2016) 121e136 123

depletion while antagonists were administered to unmanipulatedsubjects (e.g. those with normal endogenous serotonin levels). Theintent of this approach was to provide a crosscheck of the effects ofcertain receptors on ABR measures such that responses wereexamined when only one receptor type was stimulated (the sero-tonin-depletion þ agonist groups) and also when that same re-ceptor type was blocked while all other receptors were functioningnormally (antagonist groups). To activate the 5-HT1A receptoragonist, 8-Hydroxy-DPAT hydrobromide (8-OH-DPAT; TocrisBioscience, Cat#0529) was administered. This chemical has as highaffinity for the 5-HT1A subtype (Middlemiss and Fozard, 1983) aswell as a moderate affinity for the 5-HT7 receptor (Wood et al.,2000). Additional agonists included CP93129 dihydrochloride(CP93129, SigmaeAldrich, Cat#PZ0102) which is a potent and se-lective agonist of the 5-HT1B receptor, and (±)-DOI hydrochloride(DOI; Sigma Aldrich, Cat#D101) which is a potent agonist of the 5-HT2A and 2C receptors (Hoyer et al., 2002). Serotonin receptorantagonists included (S)-WAY 100135 dihydrochloride (S-WAY;Tocris Bioscience, Cat#1253) with high affinity for the 5-HT1A re-ceptor, (2R)-2-[[[3-(4-Morpholinylmethyl)-2H-1-benzopyran-8-yl]oxy]methyl] morpholinedimethanesulfonate (NAS-181; TocrisBioscience, Cat#1314) which is selective for the 5-HT1B receptor,and ketanserin tartrate (Tocris Bioscience, Cat#0908) (Hoyer et al.,2002). Though ketanserin has some affinity for both 2A and 2Creceptors, its affinity for the 5-HT2A receptor is between 10 and 30times greater than for the 5-HT2C receptor (Vollenweider et al.,

1999). 8-OH-DPAT and NAS-181 were administered subcutane-ously at a dosage of 0.5 mg/kg (Revelis et al., 1998) and 10 mg/kg(Stenfors et al., 2001), respectively. All other agonists and antago-nists were administered intraperitoneally at the following dosages:1 mg/kg for both CP93129 and DOI, 10 mg/kg of S-WAY, and 1.5 mg/kg ketanserin (Ninan and Kulkarni, 1999). Lastly, a saline solutionwas administered to a control group in order to control for theeffects of repeated handling and injections of serotonin-depletedsubjects. The quantity of injected saline was equal to the amountof serotonergic drug administered to subjects receiving pCPA in-jections, and was administered on the same timeline.

2.3. Electrode implantation

Measurement of ABRs required placement of subdermal silverwires which served as electrodes at the vertex of the head, nape ofthe neck, and on the bulla located just behind each ear. Mice werebriefly anesthetized with isoflurane fumes, and then injectedintraperitoneally with 120 mg/kg ketamine and 5 mg/kg xylazine.Surgical anesthesia was achieved as assessed by lack of response totail and toe pinch. Following induction of anesthesia, ophthalmicointment was placed over the eyes of the mouse to prevent drying.Depilatory creamwas used to clear each electrode site of hair. Eachsite was then immediately rinsed and cleaned with three applica-tions of betadine and 70% ethanol. Electrodes were constructed ofsterilized silver wire loops (26 G). The electrodes were inserted

Table 1Free-field presentation levels in dB SPL and dB SL for each test frequency.

4 kHz 8 kHz 16 kHz 32 kHz

Averagethreshold: 55 dBSPL


Averagethreshold:22.5 dB SPL


dB SPL dB SL dB SPL dB SL dB SPL dB SL dB SPL dB SL

100 45 82 50 77.5 55 71 4090 35 72 40 67.5 45 61 3080 25 62 30 57.5 35 51 2070 15 52 20 47.5 25 41 10


subcutaneously through a hypodermic needle (18 G) passedthrough a fold of skin held by forceps. Once inserted, the hypo-dermic needle was removed, leaving the silver wire electrodes inplace. The ends of the electrodes were twisted together and clippedoff approximately 3 mm from the subject's scalp. Subjects werethen treated with topical antibiotic and allowed to regain con-sciousness and mobility prior to being returned to animal quarters.Subjects were monitored daily following surgery to ensure goodrecovery and analgesics were given if indicated. No ABR measure-ments were made until at least one week following electrode im-plantation to allow for adequate healing time.

2.4. Auditory brainstem response (ABR) recordings

Prior to beginning ABR measurements, subjects were anes-thetized via brief exposure to isofluorane before receiving intra-peritoneal (IP) injections of a mixture of ketamine (120 mg/kg) andxylazine (5 mg/kg). Anesthetic level was confirmed via toe and tailpinch. Subjects were then transported to a custom-built recordingapparatus within a sound-attenuating booth. Subject head place-ment was maintained using a bite bar. Rectal temperature wasmaintained at 36.6 ± 0.2 �C using a heating pad connected to atemperature control module (FHC; Bowdoinham, ME). Anestheticlevel was checked every fifteen minutes throughout the recordingsession via tail pinch, and additional anesthetics consisting of 1/5 ofthe initial dose were given as needed to maintain a standardanesthetic plane.

ABR stimuli consisted of tone bursts centered at frequencies of 4,8, 16, and 32 kHz with an 8 m s duration including linear 0.5 msrise/fall times. Stimuli were generated at a sampling rate of333 kHz, passed through a Microstar Data Acquisition Processor(model #5216a) to an anti-aliasing filter (model FT6-2), and aprogrammable attenuator (TDT PA5; TuckereDavis Technologies,Alachua, FL) before being amplified (Samson Servo-170; SamsonTechnologies, Hauppauge, NY) and delivered to the speaker. Stimuliwere presented in free-field via a Panasonic EAS10TH100B speakerplaced at 0� azimuth and 30� above the subject at a distance ofeight inches between the center of the speaker and the center of thesubject's head. The rate of stimulus presentation was 20 Hz. Aminimum of 1024 stimulus repetitions were included in each run,with many more presentations as levels approached hearingthresholds. The majority of runs were recorded twice in order toensure good reliability of traces. The order of presentation acrossfrequencies was counterbalanced within each group such that halfthe group heard frequencies presented from low to high, and theother half of the group was presented the frequencies starting withthe highest and moving to the lowest. The total duration of eachABR recording session was approximately 1 h from presentation ofthe first stimulus to the last. The inverting electrode was located atvertex, the non-inverting electrode just ventrolateral to the rightpinna, and the ground electrode just ventrolateral to the left pinna.Response signals were sent to a pre-amplifier (Grass P5 series;Warwick, RI) powered by a regulated power supply (Grass RPS 107;Warwick, RI), bandpass filtered between 30 and 3000 Hz with a60 Hz notch filter engaged.

For experimental measures, each tone pip was presented acrossa 40 dB range in 10 dB intervals starting at the highest level anddescending to the lowest. Presentation levels varied across stim-ulus frequency due to the limitations of the mouse hearing sensi-tivity (Henry, 1979) and system constraints. The presentation levelsof each frequency in both dB SPL and dB sensation level (SL) areshown in Table 1. Hence, 4, 8, 16, and 32 kHz stimuli were presentedat levels between 70 and 100 dB SPL, 52 and 82 dB SPL, 48 and 78 dBSPL, and 41 and 71 dB SPL, respectively. In addition to these pre-sentation levels, ABR thresholds were also estimated for each

subject at each frequency with threshold defined as the lowestpresentation level which elicited identifiable ABR wavemorphology as assessed by an experienced, but not blinded, eval-uator. Determination of thresholds was conducted by first pre-senting tone pips at a clearly suprathreshold level. The presentationlevel was then reduced in 10 dB steps until a response was nolonger visually discernable. The presentation level was thenincreased by 10 dB, recordings were made, and the presentationlevel was then sequentially stepped down in 5 dB steps untilthreshold was identified. As the presentation level decreased, aminimum of 2048 sweeps were obtained for each response in orderto ensure that noise was averaged out and the best possible re-sponses were acquired. All responses to threshold and near-threshold level stimuli were repeated to ensure that true re-sponses were obtained. Threshold was defined as the lowest levelat which wave I of the ABR was discernable to an experiencedelectrophysiologist with knowledge of each subject's experimentalcondition. Wave I was selected for threshold estimates because it isgenerally has the largest amplitude in mouse and thus mostconsistently referenced for mouse ABR thresholds (Henry, 1979). A5 dB step size allowed for unambiguous distinction of presentversus absent responses, however, smaller shifts in threshold mayhave occurred which were not captured in this protocol. Stimuluslevels were calibrated using a ¼” microphone having a flat fre-quency response through 120 kHz (ACO Pacific, Model 7017) placedin the location of the subject's head during ABR recordings against apistonphone producing 94 and 104 dB (ACO Pacific, Model 511E).Stimulus presentation and response measures were obtained usingcustom software (Batlab; Dr. Donald Gans).

Auditory thresholds for each test frequency were estimated tobe the lowest level at which wave I morphology was clearlydiscernable. Auditory thresholds obtained in the current studycorresponded well to previously published work using tone-pipABRs in CBA/J mice (Henry, 1979). Waveform morphology varieswith changes in stimulus frequency and intensity such that not allwaves may be observable at every level for each test frequency,particularly wave V (Henry,1979). For this reason, sample sizes for agiven wave may vary across stimulus frequencies (e.g. wave V wasmore often observable in response to 8 kHz stimuli compared to32 kHz stimuli).

2.5. Procedure

Subject groups and experimental procedures are outlined inFig. 1. Immediately following completion of baseline ABR measures,subjects undergoing serotonin depletion (24 in total) received theirfirst injection of pCPA while still anesthetized. Subjects were thenallowed to regain consciousness and were not returned to animalquarters until they had regained mobility. For the next five days,each of these subjects was weighed daily and examined for bothbehavioral changes and proper healing and maintenance of


electrodes and surgical sites. Each was then briefly anesthetizedwith isofluorane fumes prior to receiving a dose of pCPA. Thus, eachsubject in the serotonin-depletion group received a total of sixapplications of pCPA over the course of six days beginning just afterbaseline ABR measures. On the seventh day, subjects again under-went ABR testing to assess the effects of serotonin depletion.Immediately following serotonin-depleted ABR measures, eachsubject received a dose of one of three serotonergic agonistsdescribed above. ABR measures were repeated one hour later toassess the effects of stimulating only one receptor type while otherserotonin receptors were inactive due to serotonin depletion. Thistimeframe was selected based upon previous reports indicatingthat the effects of most systemically administered serotonergicagonists and antagonists are maximal beginning approximatelyone hour following administration (Stevens et al., 2006). Subjectsremained anesthetized from the beginning of serotonin-depletedABR recordings through the administration of serotonergic ago-nists and subsequent recordings.

Twenty-four additional subjects were tested to assess the effectsof blocking one specific serotonin receptor type using a selectiveantagonist while all other receptor subtypes were operating nor-mally with normal levels of endogenous serotonin. These subjectsunderwent baseline ABR testing as described above and thenimmediately received an injection of one of the three serotoninreceptor antagonists while still under anesthesia. As with thosesubjects receiving serotonergic agonists following serotonindepletion, a period of one hour elapsed between antagonistadministration and beginning of ABR recordings to assess the ef-fects of the antagonist. Each subject received only one dose of asingle antagonist, with eight total subjects receiving each specificreceptor antagonist.

2.6. Measurement and statistical analysis of auditory brainstemresponses

Response waveforms were converted to ASCII file format andthen read into Matlab (Version 7.0, MathWorks, Natick, MA) wherethey were offline bandpass filtered from 30 to 1500 Hz. Filteredwaveforms were then imported into Excel (Microsoft Corporation,USA) where peak analysis was completed by an experienced, butnot blinded, evaluator who determined waveform peaks basedupon the highest amplitudes occurring within appropriate latencyregions of the ABR waveform (Hall, 2007). Peak amplitudes andlatencies were measured from up to 2048 stimulus presentationsper run, with many more presentations at lower sensation levels.Runs were recorded twice in order to ensure good reliability oftraces. The latencies of peaks were selected based upon the highestamplitude locations within the appropriate latency boundaries foreach component. The amplitude of each peak component wascalculated by adding the positive peak amplitude value with theabsolute value of the following trough.

Statistical analysis was completed using SPSS software (IBM,USA). In order to determine the effects of serotonin depletion onABR amplitudes and latencies, peak measures were analyzed usingrepeated-measures ANOVA including the within-subjects variables‘Condition’ (two levels: baseline and serotonin depletion, baselineand agonist, or serotonin depleted and antagonist) and ‘Presenta-tion Level’ (4 levels). ANOVAs were conducted on the latencies andamplitudes of ABR peaks obtained at each test frequency (4, 8, 16,and 32 kHz). Separate ANOVAs were run for each ABR wave sincethe sample size of each waveform varied depending upon thestimulus frequency and the particular ABR wave. The effect of se-rotonin depletion on amplitude-intensity and latency-intensityfunctions were assessed both via analysis of the interactions be-tween ‘Condition’ and ‘Presentation Level’ as well as by calculating

the slope of the latency/intensity and amplitude/intensity functionfor each individual for each ABR wave, condition, and stimulusfrequency. For each stimulus frequency, latency/intensity slopes foreach individual were assessed via repeated-measures ANOVA withthe within-subject factor Condition (two levels: baseline andpCPA). This analysis was repeated for amplitude/intensity mea-sures. For all analyses, Greenhouse-Geisser corrections wereapplied when any factor indicated violation of the assumption ofsphericity based upon Mauchly's test of sphericity. When the needfor Greenhouse-Geisser corrections was indicated, degrees offreedom were altered respectively, appearing as decimal points inTables 2e4. As expected, decreases in stimulus intensity led tosignificantly smaller amplitudes and longer latencies of all waves inresponse to all test frequencies. Thus, further analysis focusedprimarily on main effects and interactions with ‘Condition’ sincethis was the primary experimental measure of interest.

3. Results

3.1. Effects of serotonin depletion

Compared to baseline ABR measures, serotonin depletion led tosignificantly reduced latencies of most ABR peaks in response to 8and 16 kHz stimuli. This effect is shown in Fig. 2 which displays ABRrecording from a representative individual in response to the fourstimulus frequencies presented at a similar sensation level. Noticethat in response to 8 and 16 kHz, waves II through V occurredearlier in time in the pCPA condition (broken gray line) compared tothe baseline condition (solid black line). The group effects of se-rotonin depletion on ABR peak latencies are further demonstratedin Fig. 3 where ABR peaks are plotted as a function of the change inlatency between baseline and serotonin-depleted conditions inresponse to each of the four stimulus frequencies. On this plot, thatpositive latency difference values indicate decreases in latency withserotonin depletion relative to baseline while negative valuesindicate increases in latency. Though the average latency changesfor peaks in response to 4 (panel A) and 32 kHz (panel D) stimulihover around zero, average latency differences in waves II throughV in response to 8 and 16 kHz (panels B and C, respectively) pre-sentations show consistent latency decreases in serotonin-depletedconditions relative to baseline. Statistical analysis revealed that thechange in latency was significant for waves II through V and wavesIII through V in response to 8 and 16 kHz stimuli, respectively(Table 2, left panel). In contrast to the effects of serotonin depletionof peak latencies, peak amplitudes were generally unaffected byserotonin depletion with the one exception of a small but signifi-cant decrease in wave I amplitude in response to 4 kHz followingserotonin depletion.

It is possible that the daily handling of subjects in the serotonin-depletion group influenced test results. To control for this possi-bility, a separate group of eight subjects received injections of salineon the same time scale and in the same amount as subjectsreceiving injections of pCPA. The results of repeated-measuresANOVA on this control group are shown in the right panel ofTable 2. Notice that neither the amplitude nor latency of any ABRwave was significantly affected in response to any stimulus fre-quency following saline injection. This finding provides evidencethat the effects of serotonin depletion reported above are truly areflection of experimental manipulations rather than stress or fa-tigue associated with daily handling.

3.2. Serotonergic effects on wave V amplitude-intensity andlatency-intensity functions

Various studies have indicated that decreased serotonergic

Table 2Results of repeated-measures ANOVA comparing baseline and serotonin-depletion conditions (left panel) and baseline and saline treatments (right panel) across four presentation levels. Separate ANOVAswere conducted for theamplitudes and latencies of each wave at each of the four stimulus frequencies. Significant results (p � 0.05) are in bold text.

Serotonin depletion (pCPA) compared to baseline Saline compared to baseline

Wave I Wave II Wave III Wave IV Wave V Wave I Wave II Wave III Wave IV Wave V

4 kHz Latency: n¼ 23 n¼ 22 n¼ 22 n¼ 22 n¼ 16 Latency: n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 6Condition F(1,22) ¼ 0.195 F(1,21) ¼ 1.994 F(1,21) ¼ 0.021 F(1,21) ¼ 0.192 F(1,15) ¼ 3.691 Condition F(1,7) ¼ 0.256 F(1,7) ¼ 0.081 F(1,7) ¼ 0.765 F(1,7) ¼ 0.220 F(1,5) ¼ 0.094

p ¼ 0.663 p ¼ 0.173 p ¼ 0.886 p ¼ 0.666 p ¼ 0.074 p ¼ 0.628 p ¼ 0.785 p ¼ 0.411 p ¼ 0.653 p ¼ 0.771Cond*Lvl F(2.4, 53.0) ¼ 0.107 F(2.03, 42.6)¼ 1.292 F(1.68, 35.3)¼ 0.783 F(2.17, 45.6)¼ 0.626 F(1.95,

29.3)¼ 3.701Cond*Lvl F(1.57, 11.1)¼ 2.707 F(3,21) ¼ 2.615 F(3,21) ¼ 1.752 F(3,21) ¼ 0.480 F(3,15) ¼ 2.670

p ¼ 0.928 p ¼ 0.286 p ¼ 0.444 p ¼ 0.552 p¼ 0.038 p ¼ 0.071 p ¼ 0.078 p ¼ 0.187 p ¼ 0.700 p ¼ 0.085Amplitude: Amplitude:Condition F(1,22)¼ 5.163 F(1,21) ¼ 1.762 F(1,21) ¼ 0.226 F(1,22) ¼ 2.751 F(1,15) ¼ 1.395 Condition F(1,7) ¼ 0.087 F(1,7) ¼ 0.024 F(1,7) ¼ 1.499 F(1,7) ¼ 0.278 F(1,5) ¼ 0.265

p¼ 0.033 p ¼ 0.199 p ¼ 0.639 p ¼ 0.112 p ¼ 0.254 p ¼ 0.777 p ¼ 0.882 p ¼ 0.260 p ¼ 0.614 p ¼ 0.628Cond*Lvl F(1.65, 36.2)¼ 1.105 F(1.81, 38.1)¼ 2.166 F(3,63)¼ 4.429 F(1.53, 32.1)¼ 1.425 F(1.75, 29.8)¼ 1.638 Cond*Lvl F(1.48, 10.3)¼ 3.319 F(3,21) ¼ 1.383 F(3,21) ¼ 1.549 F(3,21) ¼ 0.315 F(1.82, 9.09)¼ 2.143

p ¼ 0.332 p ¼ 0.133 p¼ 0.007 p ¼ 0.253 p ¼ 0.213 p ¼ 0.087 p ¼ 0.275 p ¼ 0.231 p ¼ 0.814 p ¼ 0.1748 kHz Latency: n¼ 23 n¼ 23 n¼ 22 n¼ 22 n¼ 21 Latency: n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 8

Condition F(1,22) ¼ 0.532 F(1,22)¼ 4.952 F(1,21)¼ 8.506 F(1,21)¼ 6.181 F(1,20)¼ 5.34 Condition F(1,7) ¼ 0.771 F(1,7) ¼ 0.491 F(1,7) ¼ 0.001 F(1,7) ¼ 0.363 F(1,7) ¼ 0.089p ¼ 0.474 p¼ 0.037 p¼ 0.008 p¼ 0.021 p¼ 0.032 p ¼ 0.409 p ¼ 0.506 p ¼ 0.970 p ¼ 0.566 p ¼ 0.774

Cond*Lvl F(3, 66) ¼ 1.098 F(3, 66) ¼ 0.294 F(2.06, 43.3)¼ 2.170 F(3, 63) ¼ 1.569 F(2.33, 46.6)¼ 1.935 Cond*Lvl F(3,21) ¼ 1.793 F(1.53,10.70) ¼ 1.365

F(3,21) ¼ 1.678 F(2.04,14.27) ¼ 0.504

F(1.19, 8.34) ¼ 3.07

p ¼ 0.356 p ¼ 0.830 p ¼ 0.125 p ¼ 0.206 p ¼ 0.149 p ¼ 0.179 p ¼ 0.281 p ¼ 0.202 p ¼ 0.618 p ¼ 0.051Amplitude: Amplitude:Condition F(1,22) ¼ 0.015 F(1,22) ¼ 0.435 F(1,21) ¼ 0.112 F(1,21) ¼ 2.607 F(1,20) ¼ 0.924 Condition F(1,7) ¼ 0.293 F(1,7) ¼ 1.449 F(1,7) ¼ 0.756 F(1,7) ¼ 0.003 F(1,5) ¼ 0.700

p ¼ 0.902 p ¼ 0.517 p ¼ 0.741 p ¼ 0.121 p ¼ 0.347 p ¼ 0.605 p ¼ 0.268 p ¼ 0.413 p ¼ 0.956 p ¼ 0.430Cond*Lvl F(1.81, 39.8)¼ 0.218 F(1.56, 34.4)¼ 0.833 F(1.78, 39.2)¼ 0.448 F(2.13, 44.7)¼ 1.947 F(3, 63)¼ 3.392 Cond*Lvl F(3,21) ¼ 0.900 F(3,21) ¼ 1.624 F(3,21) ¼ 0.581 F(3,21) ¼ 0.415 F(3,21) ¼ 2.128

p ¼ 0.783 p ¼ 0.417 p ¼ 0.620 p ¼ 0.152 p¼ 0.023 p ¼ 0.458 p ¼ 0.214 p ¼ 0.634 p ¼ 0.744 p ¼ 0.12716 kHz Latency: n¼ 24 n¼ 23 n¼ 24 n¼ 23 n¼ 22 Latency: n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 8

Condition F(1,23) ¼ 0.152 F(1,22) ¼ 3.105 F(1,23)¼ 7.677 F(1,22)¼ 12.328 F(1,21)¼ 5.934 Condition F(1,7) ¼ 0.357 F(1,7) ¼ 0.086 F(1,7)< 0.001 F(1,7) ¼ 0.104 F(1,7) ¼ 0.213p ¼ 0.700 p ¼ 0.092 p¼ 0.011 p¼ 0.002 p¼ 0.024 p ¼ 0.569 p ¼ 0.778 p ¼ 1.000 p ¼ 0.757 p ¼ 0.659

Cond*Lvl F(1.34, 30.8)¼ 0.733 F(1.92, 42.2)¼ 0.296 F(3, 63) ¼ 0.882 F(1.48, 32.6)¼ 0.462 F(3,63) ¼ 1.785 Cond*Lvl F(3,21) ¼ 0.466 F(3,21) ¼ 0.272 F(3,21) ¼ 1.555 F(3,21) ¼ 2.017 F(3,21) ¼ 0.218p ¼ 0.437 p ¼ 0.736 p ¼ 0.455 p ¼ 0.576 p ¼ 0.170 p ¼ 0.709 p ¼ 0.845 p ¼ 0.230 p ¼ 0.185 p ¼ 0.883

Amplitude: Amplitude:Condition F(1,23) ¼ 0.089 F(1,22) ¼ 1.155 F(1,23) ¼ 0.087 F(1,22) ¼ 3.301 F(1,21) ¼ 0.74 Condition F(1,7) ¼ 0.0207 F(1,7) ¼ 2.333 F(1,7) ¼ 0.040 F(1,7) ¼ 0.321 F(1,7) ¼ 5.321

p ¼ 0.768 p ¼ 0.294 p ¼ 0.771 p ¼ 0.084 p ¼ 0.399 p ¼ 0.890 p ¼ 0.170 p ¼ 0.848 p ¼ 0.588 p ¼ 0.054Cond*Lvl F(1.69, 38.8)¼ 0.147 F(2.06, 45.2)¼ 1.034 F(1.93, 42.4)¼ 1.058 F(1.73, 36.3)¼ 0.635 F(2.08, 45.8)¼ 0.190 Cond*Lvl F(1.72, 12) ¼ 1.005 F(3,21) ¼ 0.688 F(1.56, 10.9)¼ 1.518 F(3,21) ¼ 0.432 F(3,21) ¼ 0.907

p ¼ 0.829 p ¼ 0.365 p ¼ 0.354 p ¼ 0.513 p ¼ 0.836 p ¼ 0.383 p ¼ 0.569 p ¼ 0.239 p ¼ 0.732 p ¼ 0.45532 kHz Latency: n¼ 22 n¼ 22 n¼ 22 n¼ 21 n¼ 15 Latency: n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 6

Condition F(1,21) ¼ 2.863 F(1,21) ¼ 1.994 F(1,21) ¼ 0.976 F(1,20) ¼ 2.216 F(1,14)< 0.001 Condition F(1,6) ¼ 1.478 F(1,6) ¼ 0.508 F(1,6) ¼ 2.470 F(1,6) ¼ 1.615 F(1,5) ¼ 1.662p ¼ 0.105 p ¼ 0.173 p ¼ 0.335 p ¼ 0.152 p ¼ 0.985 p ¼ 0.270 p ¼ 0.503 p ¼ 0.167 p ¼ 0.251 p ¼ 0.254

Cond*Lvl F(2.03, 42.6)¼ 1.172 F(1.41, 29.6)¼ 2.659 F(1.22, 25.6)¼ 1.990 F(3, 60) ¼ 0.536 F(3, 42) ¼ 0.129 Cond*Lvl F(1.35, 8.07)¼ 0.857 F(3,18) ¼ 2.265 F(3,18) ¼ 0.433 F(1.34, 8.02) ¼ 0.148 F(3,15) ¼ 1.12p ¼ 0.32 p ¼ 0.102 p ¼ 0.169 p ¼ 0.659 p ¼ 0.942 p ¼ 0.481 p ¼ 0.116 p ¼ 0.732 p ¼ 0.781 p ¼ 0.372

Amplitude: Amplitude:Condition F(1,21) ¼ 1.186 F(1,21) ¼ 2.006 F(1,21) ¼ 0.105 F(1,20) ¼ 2.55 F(1,14) ¼ 0.501 Condition F(1,6) ¼ 0.412 F(1,6) ¼ 0.047 F(1,6) ¼ 2.834 F(1,6) ¼ 0.020 F(1,5) ¼ 0.961

p ¼ 0.289 p ¼ 0.171 p ¼ 0.749 p ¼ 0.125 p ¼ 0.489 p ¼ 0.545 p ¼ 0.836 p ¼ 0.143 p ¼ 0.893 p ¼ 0.372Cond*Lvl F(1.59, 33.4)¼ 0.710 F(3, 63) ¼ 1.589 F(1.71, 35.9)¼ 1.600 F(1.75, 36.8)¼ 0.013 F(3, 48) ¼ 0.436 Cond*Lvl F(1.35, 8.07)¼ 0.907 F(3,18) ¼ 2.137 F(3, 18) ¼ 1.664 F(3, 18) ¼ 2.665 F(3,15) ¼ 3.085

p ¼ 0.468 p ¼ 0.201 p ¼ 0.218 p ¼ 0.979 p ¼ 0.728 p ¼ 0.401 p ¼ 0.131 p ¼ 0.210 p ¼ 0.079 p ¼ 0.059

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Fig. 2. ABR measures obtained during baseline (solid black) and after serotonin depletion with pCPA (broken gray) for a representative subject. Panels A through D show waveformsin response to stimulus frequencies of 4, 8, 16, and 32 kHz, respectively, presented at similar sensation levels. Notice different amplitude scales for each panel reflecting differencesin response amplitudes across stimulus frequencies and presentation levels.


neurotransmission is associated with increases in the slope of theamplitude-intensity functions of the cortical N1/P2 response(Hegerl et al., 2001), although the robustness of this phenomenonin human patients is debated (O'Neill et al., 2008). One previousstudy assessing auditory brainstem effects in depressed patientsalso reported steeper amplitude intensity functions for wave V ofthe ABR (Gopal et al., 2004). To assess the potential effects of se-rotonin depletion on wave V of the ABR across stimulus intensity,we analyzed both the interaction between ‘Condition’ and ‘Pre-sentation Level’, and also calculated the slope of each individual'slatency/intensity and amplitude/intensity functions for wave V inboth baseline and serotonin-depleted conditions. These individualslope measures were then submitted to repeated-measures ANOVAwith ‘Condition’ as the within-subjects factor with two levels(baseline and serotonin-depleted). Both of these metrics revealed asignificant effect of serotonin depletion on the latency-intensityfunction of wave V in response to 4 kHz stimuli only. The interac-tion between ‘Condition’ and ‘Presentation Level’was significant atthe level of p ¼ 0.038 (F(1.95, 29.266) ¼ 3.701; Table 2) and the maineffect of individual slope comparisons between baseline andserotonin-depleted conditions was significant at the level ofp ¼ 0.003 (F(1, 20) ¼ 11.732). The group average effect of serotonindepletion on wave V latencies in response to 4 kHz stimuli across a40 dB range of intensities is shown in Fig. 4. Compared to baselinemeasures, the slope of the latency-intensity function is shallowerfollowing serotonin depletion. This effect is driven by shorter la-tencies in response to low level stimuli in the serotonin-depletedcondition with more similar latencies occurring in response tohigher level stimuli. No significant changes were found with regardto the amplitude-intensity functions of wave V at any stimulus

frequency or on the latency-intensity functions of wave V inresponse to stimulus frequencies other than 4 kHz.

3.3. Effects of serotonergic agonists

In order to assess the effects of stimulating only select seroto-nergic receptors, three groups of subjects were given injections ofagonists selective for the 5-HT1A, 1B, and 2 receptors immediatelyfollowing ABR assessments of serotonin depletion. This experi-mental design allowed us to isolate the influence of specific sero-tonergic receptors by comparing conditions in which virtually noreceptors were activated (serotonin depletion conditions) to thoseconditions in which only select serotonergic receptors were acti-vated (agonist conditions). Repeated-measures ANOVAs wereconducted on the within-subjects variables ‘Condition’ (two levelsincluding ‘serotonin-depleted’ and ‘agonist’) and ‘PresentationLevel’ (4 levels), with separate ANOVAs conducted for latency andamplitude measures. Outcomes from these analyses are presentedin Table 3.

3.3.1. 8-OH-DPAT (5-HT1A) agonistCompared to the serotonin-depleted condition, application of

the 5-HT1A agonist 8-OH-DPAT led to significant changes in boththe amplitude and latency of many peak ABR components across awide range of stimulus frequencies (Table 3). These changes wereparticularly pervasive in responses to the lowest frequency stim-ulus, 4 kHz stimuli, at which 8-OH-DPAT significantly reduced thelatencies of waves II through V and decreased the amplitudes ofwaves II and IV. These effects on the latency and amplitude of re-sponses at 4 kHz are shown in Fig. 5, panels A and B, respectively.

Fig. 3. Average individual change in latency following serotonin depletion. For each individual, latencies measured after pCPA administration were subtracted from baseline latencymeasures. Positive values indicate shorter latencies following serotonin depletion, and negative values represent longer latencies relative to baseline measures. Error bars represent±1 SEM. * indicates significance at the level of p < 0.05. ** indicates significance at the level of p < 0.01.


Latency and amplitude changes in response to other stimulus fre-quencies were more sporadic, but displayed similar patternsparticularly with respect to amplitude changes. In response to8 kHz stimuli, all waves had generally smaller amplitudes withsignificant decreases identified at waves I, III, and IV. Significant

Fig. 4. Changes in the slope of latency/intensity function of wave V in response to4 kHz stimuli presented over a range of 40 dB measured during baseline (solid blackline) and following serotonin depletion with pCPA (broken gray line). Error barsindicate ±1 SEM.

effects in response to 16 kHz stimuli included smaller amplitudes ofwaves IV and V during 5-HT1A activation compared to serotonindepletion, with significant latency decreases and amplitude in-creases of wave II in response to 32 kHz stimuli (Table 3).

3.3.2. 5-HT1B agonist (CP93129) effectsApplication of the 5-HT1B agonist led to few significant changes

in ABR responses compared to serotonin-depleted recordings.Analysis of responses to 32 kHz stimuli indicated a significant maineffect of ‘Condition’ on the latencies of waves IV and V (Table 3)such that application of CP93129 led to shorter latencies comparedwith the pCPA condition. However, due to the small amplitude ofwave V responses to 32 kHz stimuli (see Fig. 2), the sample sizesupon which these analyses were conducted are quite small (n of 5and 4 for waves IV and V, respectively) such that any in-terpretations must be drawn with caution.

3.3.3. 5-HT2 agonist (DOI) effectsSimilar to results from the 5-HT1A receptor agonist, application

of a 5-HT2 agonist in serotonin-depleted subjects led to significantdecreases in the latencies of waves II, IV, and V in response to 4 kHzstimuli as evidenced by a significant main effect of ‘Condition’ atthis frequency (Table 3). Waves I and III also demonstrated similartrends toward significant latency decreases. Fig. 6, panel A, plots theaverage latency measured during serotonin-depleted conditions

Table 3Results of repeated-measures ANOVA comparing selective serotonin receptor agonists to serotonin-depletion conditions. Separate ANOVAs were conducted for the amplitudes and latencies of each wave at each of the fourstimulus frequencies. Significant results (p � 0.05) are in bold text.

Serotonin depletion (pCPA) compared to 5-HT1A agonist (8-OH-DPAT) Serotonin depletion (pCPA) compared to 5-HT1B agonist (CP93129) Serotonin depletion (pCPA) compared to 5-HT2 agonist (DOI)

Wave I Wave II Wave III Wave IV Wave V Wave I Wave II Wave III Wave IV Wave V Wave I Wave II Wave III Wave IV Wave V

F F F F F F F F F F F F F F F

4 kHz n¼ 8 n¼ 8 n¼ 8 n¼ 7 n¼ 6 n¼ 5 n¼ 4 n¼ 6 n¼ 6 n¼ 3 n¼ 8 n¼ 8 n¼ 7 n¼ 8 n¼ 7Latency F(1,7) ¼ 3.91 F(1,7)

¼ 15.840F(1,7) ¼ 52.320 F(1,6)

¼ 22.639F(1,5) ¼ 8.653 F(1,4) ¼ 4.704 F(1,3) ¼ 2.853 F(1,5) ¼ 0.108 F(1,5) ¼ 0.137 F(1,2) ¼ 0.142 F(1,7) ¼ 4.034 F(1,7) ¼ 6.644 F(1,6) ¼ 3.324 F(1,7) ¼ 6.260 F(1,6) ¼ 14.587

p ¼ 0.090 p ¼ 0.005 p < 0.001 p ¼ 0.003 p ¼ 0.032 p ¼ 0.096 p ¼ 0.190 p ¼ 0.756 p ¼ 0.726 p ¼ 0.743 p ¼ 0.085 p ¼ 0.037 p ¼ 0.118 p ¼ 0.041 p ¼ 0.009Amplitude F(1,7) ¼ 0.174 F(1,7)

¼ 22.381F(1,7) ¼ 0.470 F(1,6)

¼ 7.010F(1,5) ¼ 4.849 F(1,4) ¼ 1.204 F(1,3) ¼ 6.329 F(1,5) ¼ 1.346 F(1,5) ¼ 0.221 F(1,2) ¼ 1.475 F(1,7) ¼ 1.953 F(1,7) ¼ 6.584 F(1,6) ¼ 1.258 F(1,7) ¼ 0.947 F(1,6) ¼ 0.074

p ¼ 0.690 p ¼ 0.002 p ¼ 0.515 p ¼ 0.038 p ¼ 0.079 p ¼ 0.334 p ¼ 0.086 p ¼ 0.298 p ¼ 0.658 p ¼ 0.311 p ¼ 0.205 p ¼ 0.037 p ¼ 0.305 p ¼ 0.363 p ¼ 0.7938 kHz n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 6 n¼ 8 n¼ 8 n¼ 7 n¼ 8 n¼ 7Latency F(1,7) ¼ 0.452 F(1,7)

¼ 1.264F(1,7) ¼ 6.060 F(1,7)

¼ 3.832F(1,6) ¼ 0.863 F(1,6) ¼ 2.090 F(1,6) ¼ 0.025 F(1,6) ¼ 0.010 F(1,6) ¼ 1.231 F(1,5) ¼ 5.001 F(1,7) ¼ 0.630 F(1,7) ¼ 4.543 F(1,6) ¼ 3.092 F(1,7) ¼ 1.690 F(1,6) ¼ 0.792

p ¼ 0.523 p ¼ 0.298 p ¼ 0.043 p ¼ 0.091 p ¼ 0.389 p ¼ 0.198 p ¼ 0.879 p ¼ 0.925 p ¼ 0.310 p ¼ 0.076 p ¼ 0.0453 p ¼ 0.071 p ¼ 0.129 p ¼ 0.235 p ¼ 0.408Amplitude F(1,7)

¼ 23.825F(1,7)¼ 6.820

F(1,7) ¼ 0.786 F(1,7)¼ 17.939

F(1,6) ¼ 2.772 F(1,6) ¼ 0.244 F(1,6) ¼ 1.409 F(1,6)< 0.001 F(1,6) ¼ 0.519 F(1,5) ¼ 4.406 F(1,7) ¼ 1.483 F(1,7) ¼ 4.146 F(1,6) ¼ 0.307 F(1,7) ¼ 0.329 F(1,6) ¼ 0.610

p ¼ 0.002 p ¼ 0.350 p ¼ 0.405 p ¼ 0.004 p ¼ 0.147 p ¼ 0.639 p ¼ 0.280 p ¼ 0.987 p ¼ 0.498 p ¼ 0.090 p ¼ 0.263 p ¼ 0.081 p ¼ 0.597 p ¼ 0.584 p ¼ 0.46016 kHz n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 6 n¼ 7 n¼ 7 n¼ 6 n¼ 6 n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 7Latency F(1,6)< 0.000 F(1,6)

¼ 0.153F(1,6) ¼ 0.965 F(1,6)

¼ 0.653F(1,6) ¼ 1.462 F(1,5) ¼ 2.195 F(1,6) ¼ 5.165 F(1,6) ¼ 0.195 F(1,5) ¼ 0.133 F(1,5) ¼ 0.200 F(1,7) ¼ 1.455 F(1,7) ¼ 0.943 F(1,7) ¼ 0.775 F(1,7) ¼ 0.407 F(1,6) ¼ 1.063

p ¼ 1.000 p ¼ 0.709 p ¼ 0.364 p ¼ 0.450 p ¼ 0.272 p ¼ 0.199 p ¼ 0.063 p ¼ 0.674 p ¼ 0.730 p ¼ 0.673 p ¼ 0.267 p ¼ 0.364 p ¼ 0.408 p ¼ 0.544 p ¼ 0.342Amplitude F(1,6) ¼ 0.321 F(1,6)

¼ 3.715F(1,6) ¼ 0.913 F(1,6)

¼ 15.701F(1,6) ¼ 9.985 F(1,5) ¼ 0.076 F(1,6) ¼ 0.892 F(1,6) ¼ 1.569 F(1,5) ¼ 0.924 F(1,5) ¼ 6.329 F(1,7) ¼ 0.015 F(1,7) ¼ 0.419 F(1,7) ¼ 1.783 F(1,7) ¼ 0.556 F(1,6) ¼ 1.545

p ¼ 0.592 p ¼ 0.102 p ¼ 0.913 p ¼ 0.007 p ¼ 0.020 p ¼ 0.794 p ¼ 0.381 p ¼ 0.257 p ¼ 0.381 p ¼ 0.053 p ¼ 0.905 p ¼ 0.538 p ¼ 0.224 p ¼ 0.480 p ¼ 0.25432 kHz n¼ 4 n¼ 5 n¼ 5 n¼ 5 n¼ 4 n¼ 5 n¼ 5 n¼ 5 n¼ 5 n¼ 4 n¼ 8 n¼ 8 n¼ 8 n¼ 7 n¼ 2Latency F(1,3) ¼ 0.820 F(1,4)

¼ 28.560F(1,4) ¼ 0.267 F(1,4)

¼ 0.925F(1,3) ¼ 0.137 F(1,4) ¼ 0.112 F(1,4) ¼ 0.611 F(1,4) ¼ 0.598 F(1,4)

¼ 67.529F(1,3)¼ 12.835

F(1,7) ¼ 3.619 F(1,7) ¼ 3.098 F(1,7) ¼ 0.231 F(1,6) ¼ 0.008 F(1,1) ¼ 4.098

p ¼ 0.432 p ¼ 0.006 p ¼ 0.633 p ¼ 0.925 p ¼ 0.736 p ¼ 0.755 p ¼ 0.478 p ¼ 0.482 p ¼ 0.001 p ¼ 0.037 p ¼ 0.099 p ¼ 0.122 p ¼ 0.645 p ¼ 0.933 p ¼ 0.292Amplitude F(1,3) ¼ 6.736 F(1,4)

¼ 11.665F(1,4) ¼ 0.021 F(1,4)

¼ 0.427F(1,3) ¼ 6.377 F(1,4) ¼ 0.375 F(1,4) ¼ 0.527 F(1,4) ¼ 0.786 F(1,4) ¼ 0.501 F(1,3) ¼ 0.180 F(1,7) ¼ 0.191 F(1,7) ¼ 1.373 F(1,7) ¼ 0.317 F(1,6) ¼ 2.245 F(1,1) ¼ 5.030

p ¼ 0.060 p ¼ 0.027 p ¼ 0.892 p ¼ 0.549 p ¼ 0.086 p ¼ 0.573 p ¼ 0.508 p ¼ 0.425 p ¼ 0.518 p ¼ 0.700 p ¼ 0.677 p ¼ 0.280 p ¼ 0.591 p ¼ 0.185 p ¼ 0.088

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Fig. 5. Group average 5-HT1A receptor effects on ABR peaks I e V in response to 4 kHz stimuli averaged across all presentation levels. Panels A and B represent the average latencyand amplitude measures, respectively, obtained during serotonin depletion with pCPA and following application of the 5-HT1A agonist 8-OH-DPAT. Panels C and D represent theaverage latency and amplitude measures, respectively, measured during baseline and following application of the 5-HT1A antagonist S-WAY. * indicates p < 0.05. ** indicatesp < 0.01. *** indicates p < 0.001. Error bars ¼ ±1 SEM.


and following DOI application in response to 4 kHz stimuli pre-sented at 45 dB SL. From this figure, it is apparent that the latenciesof waves II, IV, and V were consistently decreased relative toserotonin-depleted conditions across subjects. The amplitude ofwave II in response to 4 kHz was also significantly decreased by theapplication of DOI (Table 3). Responses to stimulation at other testfrequencies (8, 16, and 32 kHz) demonstrated no significant maineffects of DOI application on either peak amplitudes or latencies.

3.4. Effects of serotonergic antagonists

To complement the agonist portion of the study in which onlyone specific serotonergic receptor was stimulated in the absence ofserotonin to stimulate other receptors, the effects of deactivatingthese specific receptors were investigated in a group of previouslyuntreated mice with normal levels of serotonergic activity. Hence,this paradigm allows for the assessment of responses when onlyone serotonergic receptor type is deactivated and all others arefunctioning normally, as well as when that serotonergic same re-ceptor type is the only activated receptor. Serotonergic antagonistswere administered immediately following baseline ABR measureswith additional ABR measures occurring one hour after. The effectsof antagonist application were assessed using repeated-measuresANOVAs on the within-subjects variables “Condition” (two levelsincluding “baseline” and “agonist”) and “Presentation Level” (fourlevels), with separate ANOVAs conducted for latency and amplitudemeasures. Outcomes from these analyses are presented in Table 4.

3.4.1. 5-HT1A (S-WAY) antagonistCompared to baseline measures, application of S-WAY had

minimal effects on ABR. Average response latencies and amplitudesare shown in Fig. 5, panels C and D, respectively, in response to4 kHz stimulus. This figure demonstrates the general lack of effectsof S-WAY application on either response latencies or amplitudes at

this frequency. Statistical analysis revealed no significant main ef-fect of ‘Condition’ on either the amplitude of latency of any wave atany test frequency (Table 4).

3.4.2. 5-HT1B (NAS-181) antagonistApplication of the 5-HT1B antagonist, NAS-181, revealed a

decrease in the latency of nearly all ABR peaks in response to 4 kHzstimuli. This effect is demonstrated in Fig. 6, panel B, which showsthe average measured during baseline testing and following NAS-181 application. Statistical analysis indicates that the decrease inlatency relative to baseline for responses to 4 kHz stimuli wassignificant for all waves with the exception of wave II whichshowed a clear trend toward significance (Table 4). No significantmain effects of ‘Condition’ were found on the amplitudes of anypeaks in response to 4 kHz. Effects of NAS-181 on responses tostimuli other than 4 kHz were minimal.

3.4.3. 5-HT2A (ketanserin)Blocking the action of the 5-HT2A receptor with ketanserin

revealed strong effects on response amplitudes to low and midfrequency stimuli, with no significant effects on responses to32 kHz stimuli or response latencies at any frequency (Table 4). Allsignificant amplitude effects indicated that application of ketan-serin lead to significant amplitude decreases relative to baseline. Anexample of this effect is shown in Fig. 6, panel C, which displays theaverage amplitudes measured during baseline and followingketanserin application in response to 8 kHz stimuli presented at40 dB SL. At this frequency, significant amplitude shifts were foundfor waves II, IV, and V (Table 4). Similar effects were found for wavesI and II in response to 4 kHz stimuli and waves II and IV in responseto 16 kHz stimuli (Table 4).

Fig. 6. Effects of serotonin receptor activation with DOI and deactivation using NAS-181 and Ketanserin on ABR components. Panel A demonstrates the group averagelatency in response to 4 kHz stimuli presented at 45 dB SL during serotonin depletion(pCPA) and after application of DOI. Panel B shows the group average latency beforeand after application of NAS-181 in response to the same stimulus as panel A. In PanelC, the group average amplitude is shown in response to 8 kHz stimuli presented at40 dB SL before and after Ketanserin application. Error bars ¼ SEM. * indicates p < 0.05.** indicates p < 0.01.


4. Discussion

This is the first study to explore the effects of serotonin deple-tion and serotonin receptor modulation using the ABR across awide range of stimulus frequencies and intensities. The data pre-sented here strongly support a modulatory role of serotonin onauditory brainstem processing. With regard to our initial hypoth-eses, our results indicate that 1) depletion of serotonin results inreduced ABR latencies, 2) serotonergic effects are dependent uponthe unique characteristics of specific serotonergic receptor agonistsand antagonists, and 3) the effects of manipulating serotonin andits receptors are more pronounced for lower- and mid-frequencyresponses. The diverse manner in which such effects are exertedsuggests a sophisticated role of serotonin in regulating the balanceof excitation and inhibitionwithin the auditory pathway beginningas early as the auditory nerve.

4.1. Serotonin depletion: latency and amplitude effects

Based upon data from cellular studies in the auditory brainstemdemonstrating an often suppressive role for serotonin, we hy-pothesized that reduced endogenous serotonin levels would resultin increased ABR amplitudes and decreased latencies, beginning asearly as wave I. Depletion of endogenous serotonin led to signifi-cant decreases in the latencies of most waves in response to 8 and16 kHz relative to latencies in the same individual mice beforedepletion. The fact that these changes beginwith wave II of the ABRsuggests that they begin in cochlear nucleus, although this inter-pretation must be regarded with some caution. The spiral ganglionand the cochlea receive innervation from serotonergic fibers, andthere is also evidence of serotonergic turnover in the cochleaconsistent with a functional role for serotonin (Gil-Loyzaga et al.,1997). Further, cultured spiral ganglion neurons are stronglyresponsive to serotonin receptor manipulation (Yu et al., 2013).Thus, a lack of effect of serotonin depletion on the earliest ABRwavemay suggest that effects of serotonin are not sufficiently directionalor prevalent to influence the ABR at the periphery. This interpre-tation is partially supported by the results of a previous studyassessing the effects of pCPA in prosimian primates using ABR(Revelis et al., 1998). This study revealed no significant changes inearly ABR components, with effects occurring only for wave Vwhenmeasured using similar pCPA dosages and stimulus presentationrates as the present study. However, it should be noted that theeffects they reported at wave V were in the opposite direction,reflecting increased wave V latencies after pCPA. This discrepancybetween the results of the two studiesmay reflect differences in theeffects of serotonin depletion across stimulus frequencies. Thepresent study employed tone pips of relatively narrow frequencycontent presented at low to moderate levels compared to theconsiderably higher-level broadband clicks employed in the pri-mate study (120 dB SPL). These two types of stimuli may bedominated by different cochlear regions (Eggermont and Don,1980; Abdala and Folsom, 1995), raising the possibility that spec-tral or intensity differences in the stimuli would result in differenteffects of serotonin depletion. It is also possible that the differencesbetween the studies may stem from differences between subjectspecies.

In spite of significant reductions in latency, no significantamplitude effects were found following serotonin depletion.Reduced ABR latencies are most often associated with increasedresponse amplitudes, such as occurs with increases in stimulusintensity (Picton et al., 1981). ABR amplitudes are primarilydependent upon the number of cells firing synchronously to theonset of an auditory stimulus with additional contributions ofcranial anatomy and electrode placement (Picton et al., 1981). Sig-nificant latency decreases without significant amplitude effectsfollowing serotonin depletion could thus indicate a shift in thepopulation response toward shorter response latencies withoutaffecting either the number or synchrony of responsive cells. It isalso possible that a lack of amplitude effects following serotonindepletion is related to the inherently greater variability of ABRamplitude compared to latencies which is reflected in the signifi-cantly larger relative standard deviations for ABR amplitudescompared to latencies (Musiek et al., 1984&1986; Yang et al., 1993).However, this possibility seems unlikely given that manipulation ofspecific serotonergic receptors were found to elicit significantamplitude effects. Overall, the pattern of reduced peak responselatencies with minimal changes in amplitude in the absence ofserotonin is somewhat consistent with cellular studies in variousbrainstem nuclei with regard to the intricate pattern of seroto-nergic effects in the brainstem. Rather than producing purelysuppressive or excitatory effects, serotonin depletion in the current

Table 4Results of repeated-measures ANOVA comparing selective serotonin receptor antagonists to baseline conditions. Separate ANOVAs were conducted for the amplitudes and latencies of each wave at each of the four stimulusfrequencies. Significant results (p � 0.05) are in bold text.

Baseline compared to 5-HT1A antagonist (S-WAY) Baseline compared to 5-HT1B antagonist (NAS-181) Baseline compared to 5-HT2 antagonist (ketanserin)

Wave I Wave II Wave III Wave IV Wave V Wave I Wave II Wave III Wave IV Wave V Wave I Wave II Wave III Wave IV Wave V

F F F F F F F F F F F F F F F

4 kHz n¼ 6 n¼ 7 n¼ 6 n¼ 7 n¼ 6 n¼ 7 n¼ 7 n¼ 7 n¼ 7 n¼ 2 n¼ 6 n¼ 7 n¼ 6 n¼ 7 n¼ 6Latency F(1,5) ¼ 0.040 F(1,6) ¼ 3.166 F(1,5) ¼ 4.127 F(1,6) ¼ 2.907 F(1,5) ¼ 3.270 F(1,6) ¼ 6.035 F(1,6) ¼ 4.794 F(1,6)

¼ 15.534F(1,6) ¼ 6.926 F(1,1)

¼ 29.766F(1,5) ¼ 0.016 F(1,6)

¼ 0.028F(1,5) ¼ 3.204 F(1,6) ¼ 5.534 F(1,5) ¼ 0.095

p ¼ 0.849 p ¼ 0.125 p ¼ 0.098 p ¼ 0.139 p ¼ 0.130 p ¼ 0.049 p ¼ 0.071 p ¼ 0.008 p ¼ 0.039 p ¼ 0.042 p ¼ 0.902 p ¼ 0.873 p ¼ 0.117 p ¼ 0.051 p ¼ 0.766Amplitude F(1,5) ¼ 0.419 F(1,6) ¼ 1.320 F(1,5) ¼ 0.026 F(1,6) ¼ 0.219 F(1,5) ¼ 0.979 F(1,6) ¼ 0.585 F(1,6) ¼ 1.267 F(1,6) ¼ 3.745 F(1,6) ¼ 1.127 F(1,1)

¼ 13.884F(1,5) ¼ 7.395 F(1,6)

¼ 33.731F(1,5) ¼ 2.320 F(1,6) ¼ 0.333 F(1,5) ¼ 1.795

p ¼ 0.546 p ¼ 0.294 p ¼ 0.877 p ¼ 0.656 p ¼ 0.368 p ¼ 0.473 p ¼ 0.303 p ¼ 0.101 p ¼ 0.329 p ¼ 0.167 p ¼ 0.030 p ¼ 0.001 p ¼ 0.172 p ¼ 0.582 p ¼ 0.2228 kHz n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 8 n¼ 7 n¼ 7 n¼ 6 n¼ 7 n ¼ 7 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 7Latency F(1,7) ¼ 3.671 F(1,7) ¼ 0.063 F(1,7) ¼ 2.408 F(1,7) ¼ 3.425 F(1,7) ¼ 0.287 F(1,6) ¼ 1.328 F(1,6) ¼ 0.222 F(1,5) ¼ 0.017 F(1,6) ¼ 0.048 F(1,6) ¼ 2.290 F(1,7) ¼ 1.423 F(1,7)

¼ 1.925F(1,7) ¼ 0.045 F(1,7) ¼ 0.358 F(1,6) ¼ 0.880

p ¼ 0.097 p ¼ 0.809 p ¼ 0.172 p ¼ 0.107 p ¼ 0.608 p ¼ 0.293 p ¼ 0.654 p ¼ 0.901 p ¼ 0.833 p ¼ 0.181 p ¼ 0.272 p ¼ 0.208 p ¼ 0.838 p ¼ 0.568 p ¼ 0.384Amplitude F(1,7) ¼ 1.316 F(1,7) ¼ 0.200 F(1,7) ¼ 2.089 F(1,7) ¼ 0.548 F(1,7) ¼ 0.231 F(1,6) ¼ 0.129 F(1,6) ¼ 1.755 F(1,5) ¼ 0.040 F(1,6) ¼ 1.608 F(1,6) ¼ 0.015 F(1,7) ¼ 1.308 F(1,7)

¼ 6.276F(1,7) ¼ 0.011 F(1,7)

¼ 14.792F(1,6)¼ 19.111

p ¼ 0.289 p ¼ 0.668 p ¼ 0.192 p ¼ 0.483 p ¼ 0.645 p ¼ 0.731 p ¼ 0.233 p ¼ 0.849 p ¼ 0.252 p ¼ 0.907 p ¼ 0.290 p ¼ 0.041 p ¼ 0.921 p ¼ 0.006 p ¼ 0.00516 kHz n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 7 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 7 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8Latency F(1,7) ¼ 0.070 F(1,7) ¼ 0.106 F(1,7) ¼ 0.410 F(1,7) ¼ 0.064 F(1,6) ¼ 0.261 F(1,7)< 0.001 F(1,7) ¼ 0.895 F(1,7) ¼ 1.330 F(1,7) ¼ 0.022 F(1,6) ¼ 3.712 F(1,7) ¼ 1.957 F(1,7)

¼ 1.566F(1,7) ¼ 0.122 F(1,7) ¼ 0.978 F(1,7) ¼ 0.872


¼ 7.427F(1,7) ¼ 1.760 F(1,7)

¼ 5.806F(1,7) ¼ 2.310

p ¼ 0.849 p ¼ 0.222 p ¼ 0.736 p ¼ 0.923 p ¼ 0.314 p ¼ 0.378 p ¼ 0.393 p ¼ 0.469 p ¼ 0.402 p ¼ 0.194 p ¼ 0.640 p ¼ 0.030 p ¼ 0.226 p ¼ 0.047 p ¼ 0.17232 kHz n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 7 n ¼ 5 n ¼ 5 n ¼ 5 n ¼ 5 n ¼ 5 n ¼ 2 n ¼ 8 n ¼ 8 n ¼ 7 n ¼ 8 n ¼ 5Latency F(1,7) ¼ 1.976 F(1,7) ¼ 1.828 F(1,7) ¼ 0.359 F(1,6) ¼ 0.109 F(1,4) ¼ 0.717 F(1,4) ¼ 0.093 F(1,4) ¼ 5.286 F(1,4) ¼ 1.144 F(1,4) ¼ 0.001 F(1,1) ¼ 0.001 F(1,7) ¼ 1.648 F(1,7)

¼ 0.803F(1,6) ¼ 1.447 F(1,7) ¼ 0.077 F(1,4) ¼ 0.118


¼ 2.453F(1,6) ¼ 0.584 F(1,7) ¼ 0.071 F(1,4) ¼ 0.051

p ¼ 0.233 p ¼ 0.198 p ¼ 0.342 p ¼ 0.372 p ¼ 0.448 p ¼ 0.583 p ¼ 0.042 p ¼ 0.086 p ¼ 0.219 p ¼ 0.336 p ¼ 0.302 p ¼ 0.161 p ¼ 0.474 p ¼ 0.798 p ¼ 0.832

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study may reveal reduced suppression of short latency neuronswith concomitant increased suppression of longer latency neurons.

In addition to general changes in ABR amplitudes and latencies,we investigated the possibility that low serotonin levels wouldincrease the slope of ABR amplitude/intensity and latency/intensityfunctions. Though it is currently the subject of debate, multiplestudies in both animals and humans have reported steeper slopesof amplitude/intensity functions of the cortical N1/P2 complex inindividuals with low serotonergic function (Juckel et al., 1997 &1999; Manjarrez et al., 2005; Chen et al., 2005; Gopal et al., 2005;Juckel et al., 2008; Wutzler et al., 2008; Simmons et al., 2011;Schaaf et al., 2012). In addition to these cortical studies, one studyreported similar findings of wave V of the ABR in a population ofdepressed female patients tested both on and off selective seroto-nin reuptake inhibitors (SSRIs), a class of medications which in-creases the availability of extracellular serotonin (Gopal et al.,2005). Compared to ABR responses obtained when patients werenon-medicated, SSRI use led to significantly shallower wave Vamplitude/intensity functions. In the current study, the effects ofintensity level on ABR wave V were analyzed both via assessing theinteraction between ‘Condition’ and ‘Presentation, and by analyzingthe difference in slope between baseline and serotonin depletedconditions for each individual for each wave and stimulus fre-quency. Overall, our results indicate no significant changes in theslope of the amplitude-intensity function of wave V at any testfrequency, but a significant reduction in the latency-intensityfunction for wave V at 4 kHz such that responses to lower levelstimuli elicited shorter wave V latencies in the serotonin-depletedcondition while higher level stimuli elicited similar latenciescompared to baseline measures (Fig. 4). Discrepancies between ourpresent results and those of cortical studies may be due to anumber of experimental factors, including the time course ofserotonergic manipulations, which have been proposed as animportant variable in such effects (Lee et al., 2011; Simmons et al.,2011). It is also possible that our results indicate an importantcontrast in serotonergic processing in the auditory brainstemcompared to the auditory cortex.

4.2. Serotonin receptor types

A second major hypothesis supported by our data was that ag-onists and antagonists for different serotonin receptor types wouldhave distinct suites of effects on ABR latencies and amplitudes. Thishypothesis was based on the diversity of effects of serotonin andselective serotonin receptor agonists or antagonists in multipleauditory brainstem nuclei, but the characteristic effects of agonistsand antagonists we observed could arise through multiple poten-tial pathways. These pathways include direct effects on auditoryneurons expressing serotonin receptors, combined effects onauditory neurons at different stages of auditory processing, or eventhrough effects in non-auditory brain regions projecting to auditorynuclei.

For example, the 5-HT1A receptor is expressed prominently inauditory brainstem nuclei as well as in many other brain regions(Hoyer et al., 1986; Pompeiano et al., 1992; Thompson et al., 1994).Activation of this receptor in serotonin-depleted animals caused aninteresting combination of effects: a concurrent decrease in theamplitudes and latencies of multiple waves. This is different fromwhat might be expected from a uniform decrease or increase inauditory activity, but could potentially result from effects ondifferent subsets of auditory neurons expressing the 5-HT1A re-ceptor. Within the IC, the selective activation of the 5-HT1A re-ceptor strongly decreases the responses of longer-latency neuronsto tones at the characteristic frequency, but has less of an effect onneurons with the shortest response latencies (Hurley, 2007).

Furthermore, activation of the 5-HT receptor is more likely tosuppress secondary spikes than initial spikes. In combination, theseeffects could advance ABR peaks to earlier latencies whiledecreasing amplitude. In an alternative to this scenario, the 5-HT1Areceptor is expressed by both excitatory and inhibitory neuronswithin the IC (Peruzzi and Dut, 2004). Suppressive effects of the 5-HT1A receptor on inhibitory interneurons could cause a mix ofinhibitory and disinhibitory effects apparent at the level of the ABR.Finally, the 5-HT1A receptor is an inhibitory autoreceptor withinserotonergic nuclei (Hjorth, 1993). Activation of this receptor typein the Raph�enuclei, and subsequent decrease of serotonin release inthe auditory system, could therefore also have contributed to thecombination of effects we observed.

Similar considerations apply to other types of serotonin re-ceptors. 5-HT1B receptors are located presynaptically where theyregulate the release of either excitatory or inhibitory neurotrans-mitters into the synaptic cleft (Sari, 2004; Xiao et al., 2008). Theseeffects may vary among auditory nuclei; in the IC, the effect ofactivating this receptor is consistent with disinhibition, while at theCalyx of Held in the medial nucleus of the trapezoid body, 5-HT1Breceptor stimulation reduces excitatory post-synaptic potentials(Mizutani et al., 2006). Like the 5-HT1A receptor, the 5-HT1B re-ceptor is also an inhibitory autoreceptor in some brain regions, socould potentially act to decrease serotonin release at the level ofserotonergic terminals (Engel et al., 1986). In a similar vein, phar-macological agents targeting receptors in the serotonin 2 receptorfamily excite neurons at multiple levels of the auditory brainstem(Hurley, 2006; Tang and Trussell, 2015), but also have inhibitoryeffects (Hurley, 2006). Thus, the effects of serotonin receptormanipulation that we observed are likely to represent a broad-scaleintegration of effects along the auditory neuraxis, and even be-tween auditory and non-auditory systems. Despite this impressivebreadth of potential mechanisms, the fact that manipulation ofdifferent receptor types produced different outcomes suggests thepotential for somewhat targeted effects on auditory function. Forexample, activation of the 5-HT1A receptor and block of 5-HT2receptors were two manipulations in our study that producedsignificant effects on ABR amplitude. This suggests that the 5-HT1Areceptor could be interesting to explore in the context of auditorydisorders potentially involving gain, such as hyperacusis or tinnitus(Marriage and Barnes, 1995; Nore~na et al., 1999; Simpson andDavies, 2000).

4.3. Frequency

A final hypothesis regarding the effects of serotonin depletionon ABRs was that effects would be greater in response to low fre-quency stimuli compared to higher frequency stimuli. This specu-lation was based upon immunohistochemical studies indicatingthat serotonergic innervation to lower-frequency response regionsof auditory brainstem nuclei is denser than innervation to higher-frequency response regions (Klepper and Herbert, 1991; Hurleyet al., 2002; Papesh and Hurley, 2012). Present results providelimited confirmation of this hypothesis in that serotonin depletionwas most likely to significantly affect responses to low- and mid-frequency stimuli while no effects on amplitude, latency, or in-tensity functions were found for responses to the highest frequencystimuli. This trend generally continued when assessing the effectsof serotonin receptor activation and blockade in that themajority ofsignificant effects were found in response to low- and mid-frequency stimuli (Tables 2 and 3).

This finding is especially notable, because serotonergic effectson the responses of single neurons with high characteristic fre-quencies has been documented in several studies (Hurley andPollak, 1999, 2001). Our results suggest that the largest or most


directional effects of serotonin nevertheless occur for low-frequency stimuli. Since topographic patterns of serotonergicinnervation are conserved across a range of mammalian species,particularly at the midbrain level (Thompson et al., 1994; Hurleyand Thompson, 2001), our results further suggest that low-frequency effects could likewise be a conserved feature of seroto-nergic modulation of brainstem auditory processing.

4.4. Perceptual and behavioral implications

The present study clearly demonstrated that changes inendogenous serotonin levels significantly alter temporal aspects ofthe ABR, and thus that serotonin could have considerable conse-quences for auditory processing at very early stages within theauditory pathway. Although changes in ABR latencies followingreduced serotonin levels were relatively small, these changes weremeasurable using a far-field measure such as the ABR, which in-tegrates the electrical activity of large populations of auditoryneurons. This finding suggests that either small changes in latencyor spike train timing occur in large numbers of neurons, or thatlarger changes occur in a smaller number of neurons. Although it isnot possible to distinguish among these possibilities with the ABRmeasurement, each of these would be likely to have functionallyrelevant effects, even presuming that individual neurons show verysmall shifts in latency. This is because the precise timing of excit-atory and inhibitory inputs to auditory brainstem neurons arecritical to determining selectivity of response to important acousticfeatures of vocalizations, such as direction and velocity of fre-quency changes (Andoni et al., 2007; Kao et al., 1997). Hence, evensmall changes in neural response timing may alter the brain'sability to detect important distinguishing characteristics of rapidlyfluctuating sounds such as speech. Such difficulty is likely to becompounded in noisy listening environments. Indeed, poor neuralsynchrony in the brainstem is associated with poor speech under-standing in noise (Wible et al., 2005; Song et al., 2011, 2012). Thismay partially account for why patients with disorders associatedwith poor serotonergic function report difficulty understandingspeech in noise (Gopal et al., 2004, 2005), and why auditory per-ceptions are altered by drugs affecting the serotonergic system(Parrott, 2002; Meltzer et al., 2006; Geyer and Vollenweider, 2008).We further note that ABR latency changes on a highly comparabletimescale have been previously reported as consequences of pCPAadministration (Revelis et al., 1998). Although we observed limitedchanges in the amplitudes of the ABR following manipulation ofserotonin or serotonin receptors, changes in auditory gain have alsobeen frequently-reported consequences associated with alteredserotonergic function (Norra et al., 2003; Hensch et al., 2006;Simmons et al., 2011). Because serotonergic innervation of theauditory system aswell as serotonin receptor expression are alteredfollowing noise exposure, cochlear ablation, or age-related hearingloss (Holt et al., 2005; Tadros et al., 2007; Papesh and Hurley, 2012;Smith et al., 2014), these findings are in concert with the notion thatserotonin is involved in auditory-specific disorders such as tinnitusand hyperacusis (Marriage and Barnes, 1995; Nore~na et al., 1999;Simpson and Davies, 2000; Attri and Nagarkar, 2010; Capertonand Thompson, 2011).

5. Summary

The serotonergic system is a diffuse neuromodulatory networkthrough which diverse brain regions are simultaneously influencedby the release of serotonin from the Raph�e nuclei in response toboth external stimuli and internal states (Hurley and Sullivan,2012). The present work provides one of the first extensive exam-inations of the role of serotonin and specific serotonin receptors on

far-field evoked responses in the auditory brainstem. Our resultsindicate that reductions in endogenous serotonin levels decreasepeak ABR latencies beginning at wave II and persisting throughwave V. This decrease in peak latencies is consistent with the hy-pothesis that serotonin plays a predominantly suppressive role inauditory processing such that low serotonin levels lead to increasedauditory responsiveness. We clearly showed different serotoninreceptors affect different aspects of ABR responses. Further, theeffects of serotonin depletion on ABR latencies demonstratedfrequency-specificity with more pervasive effects occurring inresponse to low and mid frequency stimuli compared to higherfrequency stimuli. Taken as a whole, our findings suggest that se-rotonin plays a powerful role in coordinating auditory activity withbehavioral states from an early level of auditory processing.

Authorship roles

Dr. Papesh was responsible for project design, implementation,analysis, and initial manuscript preparation. Dr. Hurley providedfunding and oversight of project design as well as assisting in theediting and revision of the manuscript.

Acknowledgments

This work was supported by NIDCD grant DC008963. Thesefunding sources had no role in the design, interpretation, or pub-lishing of this work. Further, there are no current or potentialconflicts of interest, either personal or financial, to be disclosedwith regard to the current work.

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