Serotonin selectively enhances perception and sensoryneural responses to stimuli generated bysame-sex conspecificsTara Deemyada,1, Michael G. Metzena, Yingzhou Pana, and Maurice J. Chacrona,b,2

Departments of aPhysiology and bPhysics, McGill University, Montreal, QC, Canada H3G 1Y6

Edited by Charles F. Stevens, Salk Institute for Biological Studies, La Jolla, CA, and approved October 23, 2013 (received for review July 24, 2013)

Centrifugal serotonergic fibers innervating sensory brain areas areseen ubiquitously across systems and species but their functionremains unclear. Here we examined the functional role of se-rotonergic innervation onto electrosensory neurons in weaklyelectric fish by eliciting endogenous release through electricalstimulation as well as exogenous focal application of serotoninin the vicinity of the cell being recorded from. Both approachesshowed that the function of serotonergic input onto electrosensorypyramidal neurons is to render themmore excitable by reducing thespike afterhyperpolarization amplitude and thereby promotingburst firing. Further, serotonergic input selectively improved neu-ronal responses to stimuli that occur during interactions betweensame-sex conspecifics but not to stimuli associated with either preyor that occur during interactions between opposite-sex conspecifics.Finally, we tested whether serotonin-mediated enhanced pyramidalneuron responses to stimuli associated with same-sex conspecificsactually increase perception by the animal. Our behavioral experi-ments show that exogenous injection and endogenous release ofserotonin both increase the magnitude of behavioral responses tostimuli associated with same-sex conspecifics as well as simulta-neously decrease aggressive behaviors. Thus, our data indicate thatthe serotonergic system inhibits aggressive behavior toward same-sex conspecifics, while at the same time increasing perception ofstimuli associated with these individuals. This function is likely tobe conserved across systems and species.

neuromodulation | neuroethology | excitability | neural coding

Animals must efficiently process natural sensory stimuli tosuccessfully interact with their environment. It has become

clear in recent years that sensory processing is not a passiveprocess but instead actively depends on behavioral context (1).Adaptive control of sensory processing is in part achieved throughneuromodulators such as serotonin (2). However, the functionalrole of serotonergic fibers emanating from the raphe nuclei in-nervating sensory brain areas remains largely unclear (3). This is inpart because these fibers make diverse patterns of connectivity(4, 5), thereby causing a wide range of effects, such as responseattenuation and gating (6, 7), as well as response enhancement(7). Thus, it is generally agreed that the function of serotonergicinput onto sensory neurons is to enhance their responses to givenstimulus features while attenuating responses to other features.As such, studies performed in model organisms well character-ized anatomically, behaviorally, and physiologically are likely tospeed progress toward a general understanding of how serotoninalters neuronal responses to natural stimuli as well as conse-quences on perception and behavior.The weakly electric fish Apteronotus leptorhynchus generates

a quasisinusoidal electric field through the electric organ dis-charge (EOD) (8). Electroreceptive neurons scattered on theskin surface monitor perturbations of this field caused by objectswith conductivity different from that of the surrounding waterand relay this information to pyramidal neurons within theelectrosensory lateral line lobe (ELL). In particular, there aretwo important categories of behaviorally relevant electrosensory

stimuli: those caused by prey are typically localized within a smallregion of the animal’s skin (9), whereas stimuli caused by con-specifics are typically diffuse and impinge on most if not all of theskin surface (8). In particular, when two fish come into contact(i.e., are within 1 m of one another), each animal will experience anamplitude modulation of its own EOD that oscillates at the dif-ference between the two EOD frequencies (i.e., a beat). Becauseof a sexual dimorphism in EOD frequency, interactions betweensame-sex conspecifics tend to give rise to low frequency beats (<30Hz), whereas interactions between opposite-sex conspecifics tend togive rise to higher frequency (30–400 Hz) beats (10). Moreover,these fish emit communication calls termed “chirps” during bothagonistic and courtship behaviors that consist of brief modulationsof their EOD. In particular, small chirps are preferentially elicitedduring encounters between same-sex conspecifics, whereas bigchirps are preferentially elicited during encounters between op-posite-sex conspecifics (10). The responses of ELL pyramidalneurons to such stimuli have been well characterized (8). Recentstudies performed in vitro have shown that serotonin renders ELLpyramidal neurons more excitable by down-regulating potassiumchannels that contribute to the spike afterhyperpolarization (AHP)(11, 12). However, the effects of serotonin on the processing ofbehaviorally relevant stimuli by pyramidal neurons in vivo and itsconsequences on behavior have not been investigated to date.Here, we used a systems level approach with behaviorally

relevant stimuli to understand the functional role of serotonergicinput onto ELL pyramidal neurons. We found that exogenous

Significance

Serotonergic innervation of sensory areas is seen ubiquitouslyacross species and systems, but its functional role remains unclearto this day. We used a systems level approach to investigate thefunctional role of serotonergic input onto electrosensory pyra-midal neurons in the weakly electric fish Apteronotus lepto-rhynchus. We found that serotonin selectively improved neuronalresponses to stimuli associated with same-sex conspecifics by in-ducing increased excitability and burst firing. Further, serotoninenhanced perception of these stimuli while simultaneouslyinhibiting aggressive behaviors. Our results provide the first evi-dence that the serotonergic system acts as a “shut up and listen”system, thereby favoring covert behavior after an aggressiveencounter together with enhanced perception of stimuli associ-ated with dominant conspecifics.

Author contributions: M.J.C. designed research; T.D., M.G.M., Y.P., and M.J.C. performedresearch; T.D., M.G.M., and Y.P. analyzed data; and T.D., M.G.M., and M.J.C. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Howard Hughes Medical Institute, Janelia Farm Research Campus,Ashburn, VA 20147.

2To whom correspondence should be addressed. E-mail: maurice.chacron@mcgill.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314008110/-/DCSupplemental.

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application of serotonin and endogenous release via electricalstimulation of serotonergic pathways both led to increased ex-citability and burst firing. Also, serotonin release enhanced py-ramidal neuron responses to the low-frequency stimuli that occurduring interactions between same-sex conspecifics as well as ago-nistic communication calls, but not to stimuli mimicking prey,opposite-sex conspecifics, or courtship communication calls. Fi-nally, we found that both endogenous release and injection ofserotonin caused enhanced behavioral responses to stimuli mim-icking same-sex conspecific interactions, while simultaneously re-ducing aggressive behavior. We conclude that the functional roleof serotonin is to enhance perception of stimuli associated withaggressive individuals, while at the same time promoting theiravoidance through covert behavior. Our results thus demonstratean important function for serotonergic input onto sensory neuronsand its consequences at the organismal level.

ResultsTo determine the functional role of serotonergic input, we exam-ined the effect of endogenous release as well as focal exogenousapplication of serotonin on electrosensory pyramidal neuron re-sponses to behaviorally relevant stimuli recorded extracellularlyand in some cases intracellularly (Materials and Methods).

Serotonin Increases Pyramidal Neuron Excitability and Burst Firing.How does serotonin alter spontaneous pyramidal cell activity?Our results show that endogenous release of serotonin (Fig. 1A,and Materials and Methods) gives rise to increased excitability(Fig. 1B). Indeed, we observed a large and significant increase inboth firing rate (Fig. 1C) and burst fraction (Fig. 1D and Mate-rials and Methods) when the stimulation electrodes were properlyplaced as verified by subsequent histology (Materials and Methodsand Fig. S1). As a control, we did not observe significant in-creases in either firing rate (Fig. 1C) or burst fraction (Fig. 1D)when the stimulation electrodes were incorrectly placed and in-stead stimulated adjacent brain areas. To verify that the observedeffects were due to serotonin release within the ELL, we insteadfocally applied serotonin in the vicinity of the recorded pyrami-dal cell (Fig. 1E, Fig. S2, and Materials and Methods). Focal se-rotonin application also caused increased excitability (Fig. 1F) asmeasured by large and significant increases in firing rate (Fig.1G) and burst fraction (Fig. 1H). Note that focal saline appli-cation did not significantly alter either firing rate (Fig. 1G) orburst fraction (Fig. 1H).

Serotonin Selectively Enhances Pyramidal Neuron Responses to StimuliAssociated with Same-Sex Conspecifics. To understand how in-creased excitability due to activation of serotonergic input canaffect pyramidal neuron responses to sensory input, we recordedtheir spiking activity before and after endogenous release ofserotonin. Our stimuli were designed to mimic two importantbehavioral contexts. First, signals mimicking conspecifics weredelivered “globally” through electrodes positioned far from andon either side of the animal (13) (Fig. 2A and Materials andMethods). Second, signals mimicking prey were delivered “locally”through a small dipole located close to the animal (Fig. 2B). Weinitially used sinusoidal waveforms of varying frequencies. Whendelivered globally, these waveforms mimic the beats that occurwhen two conspecific individuals come into close proximity to oneanother. As mentioned above, same-sex interactions tend to giverise to low frequency signals (<30 Hz), whereas opposite-sexinteractions tend to give rise to higher frequency signals.Fig. 2C shows that endogenous serotonin release significantly

increased phase locking to low- (<32 Hz) but not high-frequencyglobal stimuli. Importantly, similar results were obtained whenwe instead focally applied serotonin (Fig. S3 A and B), con-firming that these effects are primarily if not exclusively due toserotonin released within the ELL. No significant changes inphase locking were observed when applying saline focally (P >

0.13, sign-rank tests, n = 8). In contrast, endogenous serotoninrelease did not alter phase locking to local stimuli (Fig. 2D). Wefound no significant differences between the firing rates, burstfractions, or vector strength of pyramidal neurons when driven bysinusoidal local or global stimuli for all frequencies (P > 0.4, sign-rank tests), indicating that the differential effects of serotonin onlocal vs. global stimuli are most likely not due to differences inhow strong pyramidal neurons were driven by the stimuli. To-gether, these findings suggest that the function of serotonin is toselectively enhance pyramidal neuron responses to stimuli that aregenerated by interactions between same-sex conspecifics.

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Fig. 1. Serotonin increases electrosensory pyramidal neuron excitability. (A)Schematic showing how stimulation of the raphe nuclei was achieved. Shownis a dorsal view of the animal’s brain with the recording pipette and thestimulation electrode (Materials and Methods). CCb, cerebellum; EGP, emi-nentia granularis posterior; Tel, telencephalon; TeO, optic tectum. (B) Extra-cellular recording (gray) from a typical pyramidal neuron showing spontaneous(i.e., in the absence of stimulation but in the presence of the animal’s EOD)activity before (Upper) and after (Lower) raphe nuclei stimulation. The bursts ofaction potentials are shown in black. (Inset) Expanded view showing a burst ofaction potentials. (C) Population-averaged firing rate before stimulation(baseline) (Left), when the stimulation electrode was incorrectly placed (con-trol) in other brain areas (Center, n = 6), and after raphe stimulation as de-termined from histology (Right, n = 13). (D) Population-averaged burst fraction(i.e., the fraction of interspike intervals < 10ms) before stimulation (Left), whenthe stimulation electrode was stimulating adjacent brain areas (Center, n = 6),and after raphe stimulation (Right, n = 13). (E) Schematic showing the setupused to apply serotonin focally. Shown are the recording electrode that ispositioned near a pyramidal neuron and the double barrel pharmacologypipette containing glutamate and serotonin that is positioned close to thisneuron’s dendritic tree. (F) Extracellular recording (gray) showing sponta-neous activity from a typical pyramidal neuron before (Upper) and after(Lower) exogenous serotonin application. The bursts of action potentialsare shown in black. (Inset) Expanded view showing a burst of actionpotentials. (G) Population-averaged firing rate before application (Left),after saline application (Center, n = 8), and after serotonin exogenous ap-plication (Right, n = 38). (H) Population-averaged burst fraction beforeapplication (Left), after saline application (Center, n = 8), and after exog-enous application (Right, n = 38). Stars indicate statistical significance at theP = 0.05 level using a sign-rank test.

19610 | www.pnas.org/cgi/doi/10.1073/pnas.1314008110 Deemyad et al.

To test this hypothesis, we used mimics of both small (Fig. 2E,Upper traces) and big chirps (Fig. 2F, Upper traces) that occurpreferentially during agonistic interactions between same-sex con-specifics and courtship interactions between opposite-sex conspe-cifics, respectively. If our hypothesis is true, then serotonin shouldenhance pyramidal neuron responses to the former but not thelatter. Confirming our prediction, endogenous serotonin releasedecreased spiking latency and increased spiking reliability tosmall chirps (Fig. 2E). We observed similar effects when sero-tonin was instead focally applied (Fig. S3 C and D). Note that nosignificant differences in spike latency (P > 0.6, sign-rank test,n = 8) or spiking reliability (P > 0.9, sign-rank test, n = 8) wereobserved when saline was focally applied. In contrast, both en-dogenous release (Fig. 2F) as well as focal exogenous application(Fig. S3E) of serotonin did not alter responses to big chirps.Thus, our results show an important functional role for seroto-nin: to selectively enhance neural responses to stimuli that occurduring interactions between same-sex conspecifics.

Serotonin Increases Pyramidal Cell Excitability by Reducing the SpikeAfterhyperpolarization. By what mechanism(s) does serotoninenhance pyramidal cell excitability and alter responses to stimuliassociated with same-sex conspecifics? To investigate this im-portant question, we performed intracellular recordings frompyramidal cells before and after electrical stimulation of sero-tonergic pathways. As expected, electrical stimulation gave riseto increased excitability and burst firing (Fig. 3A, black spikes).Superimposing the spike shapes before and after electrical stim-ulation showed a significant decrease in the AHP (Fig. 3 B and C),thereby revealing a depolarizing afterpotential (DAP) (Fig. 3B)that promotes burst firing.

Serotonin Enhances Behavioral Responses to Stimuli Generated bySame-Sex Conspecifics. Information transmitted by neurons isonly useful to an organism if it is decoded by downstream neuronsand leads to behavior. To test whether the increased pyramidalcell responses to stimuli generated by same-sex conspecifics arebehaviorally relevant, we recorded behavioral responses of re-strained fish to mimics of such stimuli before and after serotonininjection (Fig. 4A and Materials and Methods). We focused on twobehaviors. First, the jamming avoidance response (JAR), a behav-ior by which the animal changes the frequency of its self-generatedelectric field to increase the frequency difference of jammingsignals. This complex behavior requires organization, identifica-tion, and interpretation of sensory information in the brain (14,15), and is typically used as a measure of perception in weaklyelectric fish (16). Furthermore, it is preferentially elicited by low-frequency sinusoidal stimuli that are associated with same-sexconspecific interactions. The second behavior was small chirpproduction, which was used as a control because previous studieshave shown that this behavior associated with aggression isinhibited by serotonin (17).Fig. 4 B and C show that serotonin injection both increased the

frequency excursion during the JAR and decreased aggression,as quantified by the rate at which small chirps occurred. Similarresults were seen across our dataset (Fig. 4D). We also tookadvantage of the fact that electrosensory behaviors do not re-quire movement and can thus be elicited in immobilized animals(15) and tested whether endogenous serotonin release by elec-trical stimulation would lead to similar changes in behavior.Confirming our prediction, we found that stimulation of sero-tonergic pathways also led to an increased JAR response anda significant reduction in small chirp rate (Fig. S4 A–C). Criti-cally, bilateral exogenous serotonin application in the ELL (Fig.4E and Materials and Methods) also led to a significant increase inthe JAR and a significant reduction in chirp rate (Fig. 4 F–H),thereby showing that these changes in behavior are indeed pri-marily due to serotonin directly acting on pyramidal neuron ac-tivity. Thus, these results demonstrate that increased pyramidal

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Fig. 2. Serotonin selectively increases responses to stimuli associated withsame-sex conspecifics. (A) Schematic showing global stimulation geometryfor which the stimulus was delivered through two electrodes, G1 and G2,located lateral to the animal. Global stimuli impinge upon a large fraction ofthe sensory epithelium and mimic stimuli generated by conspecifics (Mate-rials and Methods). (B) Schematic showing local stimulation geometry forwhich the stimulus was instead delivered through a small dipole locatedclose to the animal. Local stimuli impinge upon a small fraction of the sen-sory epithelium and mimic stimuli caused by prey (Materials and Methods).(C) Population-averaged vector strength values as a function of globalstimulus frequency before (gray, n = 6) and after (black, n = 6) raphe stim-ulation. (Inset) Segment from a global 4-Hz stimulus waveform (Top) andextracellular recording from a typical pyramidal neuron before (Middle) andafter (Bottom) raphe nuclei stimulation. Note the increased burst firing(black) and that the neuron phase locks much better to the stimulus afterraphe stimulation. (D) Population-averaged vector strength values as afunction of local stimulus frequency before (gray, n = 5) and after (black, n = 5)raphe stimulation. (Inset) Segment from a local 4-Hz stimulus waveform (Top)and extracellular recording from a typical pyramidal neuron before (Middle)and after (Bottom) raphe nuclei stimulation. Note the similarity between thisneuron’s activity before and after raphe stimulation. (E) Stimulus waveformdelivered globally showing a small chirp at the center (Top), raster plot(Middle) showing spike times (gray), and the first spike occurring immediatelyafter the small chirp (black) and peristimulus time histogram (PSTH) (Bottom)before (Left) and after (Right) raphe stimulation. Bar graphs show the pop-ulation-averaged normalized first spike latency (Left, n = 13) and the nor-malized SD of the first spike latency (Right, n = 13) before and after raphestimulation. (F) Stimulus waveform delivered globally showing a big chirp atthe center (Top), raster plot (Middle) showing spike times (gray), and the firstspike occurring immediately after the big chirp (black), and PSTH (Bottom)before (Left) and after (Right) raphe stimulation. Bar graphs show the nor-malized first spike latency (Left, n = 10) and its variability (Right, n = 10) beforeand after raphe stimulation. Stars indicate statistical significance at the P = 0.05level using a sign-rank test.

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cell responses mediated by serotonin do indeed lead to improvedperception at the behavioral level. Further, they show that sero-tonin can simultaneously enhance perception of and inhibit ag-gressiveness in response to stimuli that occur during interactionsbetween same-sex conspecifics.

DiscussionWe investigated how serotonergic inputs onto electrosensorypyramidal neurons alter their responses to sensory input and itsconsequences on behavior. We found that activation of seroto-nergic pathways led to increased pyramidal neuron excitabilityand burst firing by reducing the AHP. Further, such activationselectively enhanced neuronal responses to low-frequency sinu-soidal as well as agonistic communication signals that are asso-ciated with same-sex conspecifics. We also found that endogenousrelease of serotonin can inhibit behavioral responses associatedwith aggression while at the same time enhancing behavioral re-sponses to stimuli associated with same-sex conspecifics. Thus, ourresults demonstrate an important function for serotonergic inputsonto electrosensory pyramidal neurons as well as the underlyingmechanism by which it is achieved.Our results have shown that serotonin increases excitability

in vivo by reducing the AHP and thereby promoting burst firing.The similar effects observed for both endogenous release andfocal application suggest that serotonin acts directly on seroto-nergic receptors located on pyramidal neurons. Further, the re-markable similarity between the effects of serotonin in vivo andin vitro (11) strongly suggests that the mode of action found inthe latter (i.e., down-regulation of potassium currents that con-tribute to the AHP) also applies to the former. Previous resultshave shown that electrosensory pyramidal neurons can fire burstsof action potentials through a somatodendritic interaction in-volving a DAP (18), which is similar to that seen in hippocampalpyramidal neurons. Such burst firing in electrosensory pyramidalneurons is extensively regulated (19, 20). In particular, the strongAHP seen in vivo counteracts the DAP and generally opposes

burst firing (13). However, recent studies have shown that smallchirps can elicit burst firing (21). Together with previous resultsshowing that burst firing in ELL pyramidal cells codes for low-frequency stimuli (22), our results suggest that the DAP-mediatedburst firing seen in pyramidal neurons primarily serves to signalthe occurrence of stimuli associated with same-sex conspecifics.Why does serotonin selectively affect pyramidal neuron respon-

ses to stimuli associated with interactions between same-sex con-specifics? Pyramidal neurons receive massive projections fromhigher brain centers (23) whose function is to attenuate responsesto low-frequency (<32 Hz) global stimuli by providing a negativeimage of the stimulus (24). It is important to note that neither localstimuli (24, 25) nor high-frequency (>32 Hz) global stimuli (26)elicit this feedback input and that small chirp stimuli typically occuron top of low-frequency beats (21). Further, previous findings haveshown that serotonergic fibers are mostly colocalized with feedbackinput onto pyramidal neurons (11). Therefore, we propose thatserotonin selectively enhances responses to stimuli that occur dur-ing interactions between same-sex conspecifics because only theseelicit feedback input onto pyramidal neurons that can be altered byserotonin in the first place.It is likely that the mechanisms by which serotonin alters py-

ramidal neuron activity will be found in other systems. This isbecause: (i) many neurons display burst firing similar to ELLpyramidal neurons (27) and such burst firing also encodes similarstimulus features (28); (ii) potassium channels in weakly electricfish display over 90% functional homology with their mammaliancounterparts (29); and (iii) reduction of potassium conductancesby serotonin has been shown to decrease first spike latency andincrease excitability in mammalian sensory neurons (30), therebygiving rise to greater signal detection (31).Whereas our results have shown that changes in pyramidal

neuron activity due to serotonin are sufficient to elicit changes inbehavior, it is likely that other brain areas contribute to this ef-fect under natural conditions. Indeed, other higher order sensoryareas have been shown to receive serotonergic innervation (32);for example, neurons within the midbrain torus semicircularis(TS) that receive synaptic input from pyramidal neurons receiveserotonergic innervation. Whereas previous studies have shownthat TS neurons can respond selectively to behaviorally relevantstimuli such as chirps (33), the effects of serotonin on theseresponses are unknown. It is likely that serotonin will selectivelyimprove the responses of some TS neurons, whereas reducingthose of others, as observed in homologous midbrain areas (3).However, based on the results presented here, we might expectthat only the TS neurons that respond selectively to either smallchirps or low-frequency beats would have their responses en-hanced. Further studies are needed to understand how serotoninalters the responses of TS neurons.Finally, both endogenous release as well as injection of sero-

tonin led to increased perception of low-frequency stimuli thatoccur during interactions between same-sex conspecifics whilesimultaneously inhibiting aggression. Thus, we propose that theserotonergic system acts as a “shut up and listen” system, therebyfavoring more cautious behavior after an aggressive encounter to-gether with enhanced perception of stimuli associated with domi-nant individuals. Indeed, serotonin is one of the main modulatorysystems of aggression (34). It is well known that serotonin will de-crease aggressive behaviors across species (35) as well as promoteavoidance and escape from dangerous situations (36). Importantly,our results provide a functional role linking perception to behaviorfor the commonly observed larger levels of serotonin in submissiveindividuals with respect to those observed in dominant ones (37).Specifically, we propose that these larger levels of serotonin selec-tively enhance sensory neuron responses to stimuli associated withaggressive conspecifics, thereby leading to enhanced perception andbetter avoidance by more submissive individuals.In summary, we have shown that the function of serotonergic

inputs onto ELL pyramidal cells is to selectively enhance their

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Fig. 3. Serotonin alters pyramidal neuron excitability by reducing the spikeafterhyperpolarization (AHP). (A) Intracellular recording (gray) from a typi-cal pyramidal neuron before (Upper) and after (Lower) endogenous sero-tonin release with the bursts shown in black. (B) Average action potentialwaveforms from this same neuron before (gray) and after (black) endoge-nous serotonin release showing a reduced AHP (arrowhead) as well asa depolarizing afterpotential (DAP, arrow). (C) Measured AHP (Materialsand Methods) before (Left, n = 8) and after (Right, n = 8) endogenous se-rotonin release.

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responses to stimuli associated with same-sex conspecifics througha reduction of the AHP, which leads to their enhanced perceptionat the organismal level. This important and function is likely to beshared among organisms, given that the serotonergic system isremarkably conserved across vertebrates (38).

Materials and MethodsAnimals.We used 58 weakly electric fish A. leptorhynchus of both sexes in thepresent experiments. Animals were purchased from tropical fish suppliersand were exclusively used in these experiments. Fish were acclimated tolaboratory conditions and housed in groups of 8–10. Water conductivity wasbetween 400 and 800 μS, the pH was maintained between 6.8 and 7.2, andthe temperature was kept between 27 and 29 °C (15). McGill University’sAnimal Care Committee approved all procedures.

Surgical procedures were explained in detail previously (13). Briefly, 0.1–0.5 mg of tubocurarine (Sigma) was injected intramuscularly to immobilizethe fish. The fish was then transferred to a recording tank and respirated viaa mouth tube at a flow rate of 10 mL/min. To stabilize the head duringrecording, a metal post was glued to the exposed area of the skull, rostral tothe opening. We drilled a small hole of ∼2 mm2 over the cerebellum and theELL area, caudal to the border between hindbrain and midbrain in order toaccess the pyramidal neurons. In some experiments, an opening in themidline, 1 mm rostral to border between hindbrain and midbrain was madeto gain access to the ipsilateral serotonergic fibers originating from raphenucleus for electrical stimulation.

Recordings.We usedwell-established techniques to record extracellularly andintracellular from pyramidal cells within the lateral segment (LS) of the ELL(13), which receive the greatest amount of serotonergic innervation (11). Weused CED 1401-plus hardware and Spike II software to detect both extra-cellular and intracellular spikes at a resolution of 0.1 ms (CambridgeElectronic Design).

Pharmacology. Glutamate (Sigma) and serotonin (Sigma) were dissolved insaline for application. Drug application electrodes were two-barrel KG-33glass micropipettes (OD 1.5 mm, Garner Glass Co.) pulled by a micropipettepuller (David Kopf Instruments) to a fine tip and subsequently broken toattain a tip diameter of ∼10 μm as done previously (13). The two barrels wereused for separate application of serotonin (1 mM) and glutamate (1 mM).During recordings, we first used excitatory responses to glutamate applica-tion to verify that we were in the correct location (Fig. S2). Serotonin wasthen applied as done previously (13). Note that previous studies have shownthat saline applied in this manner does not significantly alter pyramidalneuron activity (39).

Stimulation. As the electric organ discharge of A. leptorhynchus is neuro-genic, it is not affected by immobilization with curare-like drugs. All stimuliconsisted of amplitude modulations (AMs) of the animal’s own EOD andwere produced by applying a train of sinusoidal waveforms to the fish. Eachsinusoid was triggered at the zero crossing of each EOD cycle and hada period slightly less than that of the EOD waveform; hence the trainremains synchronized to the animal’s discharge and, depending on its po-larity, either adds to or subtracts from the animal’s own discharge. A mod-ulation waveform was then multiplied with the train of sinusoidal waveforms(MT3 multiplier; Tucker Davis Technologies) and the resulting signal was firstisolated from ground (A395 linear stimulus isolator; World Precision Instru-ments) before being delivered using either global or local stimulation geom-etry. For global stimulation, signals were delivered through pairs of chloridizedsilver wire electrodes positioned 15 cm away from the fish in either side of therecording tank. In contrast, for local stimulation, we used a small local dipoleelectrode that was located 1–3 mm from the skin. The intensities of local andglobal stimuli were similar to those used previously and were adjusted such asto give rise to similar changes in EOD amplitude as measured by a small dipoleclose to the animal’s skin (40). Four different types of stimuli were applied:sinusoids with frequencies 1, 2, 4, 8, 16, 32, 64, 128, and 256 Hz that wereapplied either globally or locally, as well as small and big chirps that wereapplied globally. All stimuli were generated as done previously (33).

Electrical Stimulation of Serotonergic Fibers onto ELL Pyramidal Neurons. Abipolar tungsten electrode was positioned over the serotonergic fiberstraveling to ELL, 1 mm rostral to T0 at the midline level of the brain surfaceand 3.5 mm in depth. The electrical stimulating electrode was positioned sothat the stimulation affected the chirps produced by fish. Stimuli consisted of10 pulses (200–300 pA) at 40 Hz with each pulse lasting 0.3 ms.

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Fig. 4. Serotonin simultaneously enhances behavioral responses to stimuligenerated by same-sex conspecifics and inhibits aggressive behaviors. (A)Schematic of the experimental setup in which a fish is restrained in a tube(chirp chamber) in an otherwise empty tank. The animal’s electric field ismonitored by a pair of electrodes E1, E2 (straight lines) while the stimulus (i.e.,the EOD AM; Materials and Methods) is delivered using global stimulationgeometry S1, S2 (spirals). (B, Left) Electric organ discharge (EOD) frequency asa function of time before (blue) and after (red) saline injection. The shadedregion shows the time when the stimulus was on. Arrows indicate the JARmagnitude. (Right) EOD frequency as a function of time before (blue) andafter (red) serotonin injection. (C) EOD spectrogram (i.e., EOD power spectrumas a function of time) with the vertical bands corresponding to the blackvertical bars representing chirps before injection (Left), after saline injection(Center), and after serotonin injection (Right). (D, Left) Chirp rate before in-jection (Left, n = 6), after saline injection (Center, n = 6), and after serotonininjection (Right, n = 6). (Right) JAR magnitude before injection (Left, n = 6),after saline injection (Center, n = 6), and after serotonin injection (Right, n =6). Stars indicate statistical significance at the P = 0.05 level using a sign-ranktest. (E) Schematic of the experimental setup used for bilateral exogenousserotonin application in the ELL. Two electrodes containing serotonin wereplaced in each ELL (Materials and Methods). (F) EOD frequency from an ex-ample specimen as a function of time before (blue) and after (red) serotonininjection. (G) Chirp rate before application (Left, n = 6), after saline application(Center, n = 6), and after serotonin application (Right, n = 6). (H) JAR mag-nitude before application (Left, n = 6), after saline application (Center, n = 6),and after serotonin application (Right, n = 6). Stars indicate statistical signifi-cance at the 0.05 level using a sign-rank test.

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Histology. Following completion of recordings, fish (n = 17) were deeplyanesthetized with Tricaine methanesulfonate (MS-222). The brain was thenextracted and moved to paraformaldehyde (0.04 g/mL) at 4 °C for 72 h andthen transferred to ethanol (0.592 g/mL). Prussian blue staining for iron wasperformed to localize stimulation sites, as described by previous studies (41).Brain slices of 5-μm thickness were made and immersed for 20 min in a so-lution of 0.22 g/mL hydrochloric acid and 0.185 g/mL potassium ferrocya-nide. After washing three times in distilled water, slices were counterstainedwith Nuclear Fast Red for 5 min and rinsed two times in distilled water. Next,they were dehydrated through ethanol (0.592 and 0.789 g/mL) and werecleared in 860 g/mL xylene solution (for 3 min, twice). We then verified theexact location of the electrical stimulation (Fig. S1).

Data Analysis. Burst fraction was computed as the fraction of interspikeintervals that are less than 10 ms (11, 13). Average action potential wave-forms were computed to measure the AHP following each action potential(42). The vector strength was computed as done previously (33). The latency(in milliseconds) of the first spike generated by neurons after the beginningof small and big chirp stimuli was measured and the SD of these values wasused as a measure of the variability of this latency. All quantities arereported as mean ± SE.

Behavior. For some behavioral experiments, animals were placed in a “chirpchamber” (15). The animals were injected i.m. with 0.1 mL of saline solution.Additionally, the animals were injected i.m. with 0.01–0.06 mL of 10 mMserotonin hydrochloride solution to achieve a dose of 10 μg/1 g of bodymass. Another set of behavioral experiments was performed in immobilizedanimals in response to 4-Hz sinusoidal AMs of the EOD after either bilateralinjection of serotonin or saline in the ELL molecular layer (Fig. 4E) or afterendogenous release of serotonin following electrical stimulation of seroto-nergic pathway (see above) (Fig. S4).

Sinusoidal waveforms with frequency of 4 Hz below the animal’s baselineEOD frequency and with intensity of 1.39 mV/cm with duration at least 15 swere presented. Previous studies have shown that such stimuli will reliablyelicit JAR and chirp responses in A. leptorhynchus (15). The stimuli wererepeated five times with a rest period of 50 s between each presentation.The EOD frequency was estimated as before (15). The JAR magnitude wasdefined as the maximum frequency elicited during stimulation minus thebaseline (i.e., without stimulation) value.

ACKNOWLEDGMENTS. The authors thank Dr. Leonard Maler for usefuldiscussions and Dr. Ana Giassi for assistance with histology. This work wassupported by grants from the Canadian Institutes of Health Research andthe Fonds de recherche du Québec - Nature et technologies (to M.J.C.).

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