REVIEW

Philip K. Stoddard Æ Harold H. Zakon

Michael R. Markham Æ Lynne McAnelly

Regulation and modulation of electric waveforms in gymnotiformelectric fish

Received: 7 February 2005 / Revised: 10 November 2005 / Accepted: 26 December 2005 / Published online: 26 January 2006� Springer-Verlag 2006

Abstract Weakly electric gymnotiform fish specialize inthe regulation and modulation of the action potentialsthat make up their multi-purpose electric signals. Toproduce communication signals, gymnotiform fishmodulate the waveforms of their electric organ dis-charges (EODs) over timescales spanning ten orders ofmagnitude within the animal’s life cycle: developmen-tal, reproductive, circadian, and behavioral. Rapidchanges lasting milliseconds to seconds are the result ofdirect neural control of action potential firing in theelectric organ. Intermediate-term changes taking min-utes to hours result from the action of melanocortinpeptides, the pituitary hormones that induce skindarkening and cortisol release in many vertebrates.Long-term changes in the EOD waveform taking daysto weeks result from the action of sex steroids on theelectrocytes in the electric organ as well as changes inthe neural control structures in the brain. These long-term changes in the electric organ seem to be associatedwith changes in the expression of voltage-gated ionchannels in two gene families. Electric organs expressmultiple voltage-gated sodium channel genes, at leastone of which seems to be regulated by androgens.Electric organs also express multiple subunits of theshaker (Kv1) family of voltage-gated potassium chan-nels. Expression of the Kv1 subtype has been found tovary with the duration of the waveform in the electricsignal. Our increasing understanding of the mecha-nisms underlying precise control of electric communi-cation signals may yield significant insights into thediversity of natural mechanisms available for modifying

the performance of ion channels in excitable mem-branes. These mechanisms may lead to better under-standing of normal function in a wide range ofphysiological systems and future application in treat-ment of disease states involving pathology of excitablemembranes.

Keywords Androgens Æ Electrogenesis ÆPhenotypic plasticity

Introduction

Moment to moment an animal’s behavior is influencedby the stimuli it encounters. Responses to environmentalstimuli are frequently not instantaneous, but vary over awide variety of timescales. In most species, behavior isexpressed within a circadian cycle and suites of behav-iors may be further partitioned within a particular cir-cadian phase. Over longer timescales, animals showseasonal changes in behavior. This is most obvious inseasonally breeding species where reproductive behav-iors develop and are expressed over many days or weeks.

In keeping with the time courses of these influenceson behavior, neurotransmitters, neuromodulators, andhormones have different modes of action in which theireffects may be rapid (seconds or minutes), intermediate-term (tens of minutes to hours), or long-lasting (days toweeks) (Fig. 1). While much is known about the actionsof these agents on the cellular and molecular levels, theweak link lies in our understanding of how cellularevents influence behavior. Electric fish are superb ani-mals in which to study this problem. Their electricalbehaviors are simple and easily quantified. The circuitsgenerating these behaviors are known, simple, hierar-chically organized, and largely amenable to electro-physiological and molecular analysis. Because thesebehaviors are in the currency of the nervous sys-tem—electricity—it is easy to generate realistic andtestable hypotheses about how behavior is generatedand regulated at the level of membrane biophysics. In

P. K. Stoddard (&) Æ M. R. MarkhamDepartment of Biological Sciences,Florida International University, 11200 SW 8th St,Miami, FL 33199, USAE-mail: stoddard@fiu.edu

H. H. Zakon Æ L. McAnellySection of Neurobiology, Patterson Labs,University of Texas Austin,Austin, TX 78712, USA

J Comp Physiol A (2006) 192: 613–624DOI 10.1007/s00359-006-0101-1

this paper, we discuss how behaviorally relevant varia-tions in the waveform of the electric organ discharge(EOD) of the electric fish are modulated by peptide

neuromodulators and steroid hormones, and how theseagents modulate ion currents to cause the changes inbehavior. Much of the work on hormonal regulation ofthe EOD centers on two species of South Americangymnotiform fish, Sternopygus macrurus, the gold-linedor black knife fish, and Brachyhypopomus pinnicaudatus(Hopkins 1991), the feather-tail knife fish or ‘‘pinni;’’ sowe focus our review on these species.

The EOD-generating circuitry

EODs are triggered by a midline medullary nucleuscalled the pacemaker nucleus. Within the nucleus are50–100 intrinsically active pacemaker neurons. Pace-maker neurons make electrotonic and chemical synapseson each other, and on a second group of neurons withinthe pacemaker nucleus called relay cells (Bennett et al.1967; Elekes and Szabo 1981). Pacemaker neurons arespontaneously active and fire at high frequencies withhighly regular rates (hundreds of Hz) in the wave fish,and fire at slower and less precise rates in the pulse fish(tens of Hz; Spiro 1997). These oscillatory neuronsmaintain their rhythmicity even when removed from afish and placed in a slice chamber (Dye 1991; Smith andZakon 2000).

The extensive interconnections among and betweenthe pacemaker and the relay neurons cause them to firesynchronously. The relay cells project out of the nucleusand down the spinal cord to synapse on the specializedspinal motor neurons called electromotoneurons. Theseinnervate the muscle-derived cells of the electric organ,the electrocytes. The electrocytes are usually arrangedinto rows in which they are oriented in the same axis andensheathed in electrically resistive connective tissue. Theresistive sheathing channels current along the axis of theelectric organ, out into the water, and back into the otherend of the electric organ (Bennett and Grundfest 1959;Bennett 1961; Bell et al. 1976).

The waveforms of EODs differ among species in thenumber and polarity of their phases and by differences inthe amplitude and duration of each phase. The numberof phases and their polarities are determined by fixedmorphological parameters, such as the site of innerva-tion, whether individual electrocytes have single ormultiple electrically excitable surfaces, the geometry ofthe electrocytes, and their location in the body (reviewsBass 1986; Caputi 1999; Hopkins 1999). Variation inwaveform within a species, such as sex differences orindividual differences, depends on modifiable physio-logical properties of the electrocyte such as the durationand amplitude of the action potentials (APs) generatedby each face (see below).

The simplest EOD is a monophasic pulse, such asthat produced by S. macrurus, which is generated byan electrocyte with one electrically excitable face andanother face that is electrically inexcitable (Keynesand Martins-Ferreira 1953; Bennett 1961). Upondepolarization by synaptic input, the excitable face

Fig. 1 EOD waveforms change over timescales spanning ten ordersof magnitude. Shown here are three general classes of waveformplasticity in the EOD of Brachyhypopomus pinnicaudatus. Devel-opmental changes and sexual differentiation are the slowest, drivenby growth factors and sex steroid hormones. Circadian and rapidsocial changes are intermediate, driven by monoamines andmelanocortin peptides. Complex social signals are the fastest,driven by direct neural control

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fires an AP during which a positive current (Na+

ions) enters that face and a positive current (probablyK+ ions) exits the opposite face through leak chan-nels. In other words, if the posterior face of an elec-trocyte is excitable, then current flows in that face andout the anterior face; the net positive current from allthe simultaneously active electrocytes flows headwardalong the length of the electric organ into the water,and back into the fish’s tail. This results in a head-positive pulse.

Both faces of the electrocyte are excitable in thosespecies that produce a biphasic discharge, such as B.pinnicaudatus. First, the innervated face fires an APduring which the current flows toward the head. Then,the flow of current through the other membrane depo-larizes it causing an AP to be generated with the currentflowing toward the tail. The alternate flow of the currentheadward and then tailward gives the EOD its biphasicshape (Bennett 1961).

Some species, the ‘‘wave fish,’’ generate sine wave-likeEODs. Wave-type EODs are essentially pulse-typeEODs generated at an extremely regular rate. If thedurations of the EOD pulses are about the same as thoseof the inter-pulse intervals, the EOD will be periodic and

nearly sinusoidal. In wave fish of the family Sterno-pygidae, the EOD pulse is monophasic because thesimple electrocytes have single active faces. The fre-quency of a wave-type EOD is determined by the firingfrequency of the pacemaker nucleus. However, for thewaveform to approximate a sine wave, the EOD pulseduration must also vary with the pulse rate. So, forexample, the EOD frequency of S. macrurus varies from50 to 200 Hz and EOD pulse duration varies from 14 to4 ms (Fig. 2). These two parameters co-vary so that thefish with the lowest EOD frequency has the longest pulse(Mills and Zakon 1987, 1991). These two independentparameters may be manipulated by hormonal modula-tion (see below).

The EOD as a communication signal

The EOD serves as a multi-purpose system: it is used tosense objects around the fish and also for communicationamong conspecifics. EOD parameters are sexuallydimorphic and often individually distinct. Sex differencesin the waveform appear related exclusively to commu-nication. Though male and female Brachyhypopomus

Fig. 2 EOD-generatingcircuitry in Sternopygus. a TheEOD is triggered by neurons ofthe pacemaker nucleus in thehindbrain, which activate theelectric organ via b descendingprojections to spinalelectromotor neurons. EachEOD pulse is the summation ofthe action potentials of the cellsof the electric organ, theelectrocytes. c Each actionpotential in the pacemakernucleus is followed by a pulsefrom the electric organ. In awave fish, such as Sternopygus,the pacemaker neurons offemales discharge at a high rateand their electrocytes produce abrief action potential togenerate a sinusoidal EOD at ahigh frequency. d Thepacemaker neurons in males, onthe other hand, fire at lowerrates, and the EOD pulse is oflonger duration to preserve thesinusoidal nature of the EOD(McAnelly and Zakon 2000)

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have strikingly different waveforms, limited testingshows their acuity in electrolocation to be identical(Fig. 3; Olman 2001).

One commonality of pulse and wave fish is that theEOD pulses of mature males are frequently longer thanthose of mature females or juveniles. In some species ofpulse fish with biphasic EOD pulses, both phases of themale EOD pulse are longer in duration than the equiva-lent phases in females of the species; while in others, onlyone phase of the EOD is sexually dimorphic. The firstphase of the EOD pulse is similar in duration in male andfemale B. pinnicaudatus, but the second phase is longer in

males than in females (Hopkins et al. 1990; Hopkins1991). As discussed above, the EOD pulse also varies induration in the wave fish Sternopygus. Mature malesdischarge at low frequencies and have long EOD pulses,while mature females discharge at higher frequencies andhave short EOD pulses. Behavioral studies indicate thatfish discriminate between male and female EODs of theirspecies. For example, male Sternopygus will court dipoleplayback electrodes emitting synthetically generated fe-male EOD mimics (Hopkins 1974). Likewise, femaleB. occidentalis responded differentially to single periodsine waves that match male-typical and female-typicalspectral peaks (Shumway and Zelick 1988). Eigenmanniahave been conditioned to make similar discriminations insynthetic waveforms (Kramer and Zupanc 1986).

Rapid changes in the EOD

Rapid changes in the EOD waveform result when themedullary pacemaker nucleus drives the EOD fasterthan the electrocytes in the peripheral electric organs cancontinue to produce full APs. At these times, the EODsmay diminish in amplitude or fail entirely. Theseamplitude modulations make up the basis of dynamiccourtship signals (review Stoddard 2002).

Intermediate-term changes in the EOD

The genus Brachyhypopomus appears to be a specialist inintermediate-term modification of its EOD waveformoccurring over the course of minutes to hours. Matureindividuals of both sexes display day–night changes inthe amplitude of the EOD and the duration of the sec-ond phase (Hagedorn 1995; Franchina and Stoddard1998), the same features that are sexually dimorphic.These rhythms are much larger and more consistentlyexpressed by males (Franchina and Stoddard 1998; Silvaet al. 1999). The day–night changes are true circadianrhythms that free-run under conditions of constant lightor constant dark, constant temperature, and randomfeeding (Fig. 4; Stoddard et al. 2003; P.K. Stoddardet al., unpublished data). Increases in amplitude andsecond phase duration of the EOD waveform(enhancement of the sexual dimorphisms) generally be-gin in the late afternoon. By sunset when the fish becomeactive, the changes are in rapid swing. An hour afterdark when the initiation of courtship is most frequent,the EODs are fully enhanced. After midnight, the EODcycle begins to wane.

In addition to circadian oscillation, EODs of Brachy-hypopomus of both sexes undergo rapid enhancement inresponse to potentially aggressive social contacts (Fig. 5).When a conspecific is introduced to another individual’sresting place, the EODs of both individuals undergo rapidenhancement, first evident in 5–10 min, and increasingover the next 45 min (Franchina et al. 2001; Stoddardet al. 2003). After competing for a resting place, winners

Fig. 3 Active electroreception thresholds of B. pinnicaudatus formoving objects are not affected by amplitude or second phaseduration of the EOD (Olman 2001). To determine object detectionthresholds, fish were placed in a screen tube located behind a rigidbut porous polyethylene barrier permeable to electric signals, butimpermeable to mechanical waves that might activate the lateralline system. A 3 cm square sheet of plastic 1 mm thick was movedat 10 cm s�1 along a 50 cm track parallel to the fish’s head–tailaxis, at decreasing distances lateral to the fish. The fish’s EOD ratewas monitored throughout. Plots show the change in maximumdischarge rate from that during a control period immediately priorto the test run. The fish increased its discharge rate when it noticedthe object, a reflex in pulse fish known as the novelty response. Thedetection threshold was considered to be the greatest distance witha sharp increase in EOD rate. EOD waveforms of subjects plottedin boxes sized to that of the second fish, male 14. Neither loweramplitude (e.g., male 12) nor lower second phase duration (e.g.,male 14 after 1 week isolation) affected the object detectionthreshold. Insensitivity to waveform differences should not besurprising if one realizes that the EOD is variable primarily at thetail whereas the EOD varies little at the head (Stoddard et al. 1999),and the head harbors the most sensitive electroreceptors (Yagerand Hopkins 1993) at the greatest density (Szabo 1974)

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retain more masculine EOD characteristics than losers(Hagedorn and Zelick 1989).

When B. pinnicaudatus dyads interact at night, one orboth may show rapid EOD enhancement (Franchinaet al. 2001) but not always. The change depends on theprior state of the individuals, their sex, and their com-petitive status. Generally dominant males show andretain the greatest increase in EOD parameters (S. Alleeand V.L. Salazar, unpublished data), but when animalsare allowed to interact through electrically transparent

mesh the responses are variable: in some instances asmaller resident male enhances his EOD while a largermale, introduced to the tank for a few hours, reduces hisEOD. We have seen similar effects in limited trials withSternopygus. Studies are currently underway to clarifythe nature of the responses to individuals of differentcompetitive status.

Social isolation of sexually mature male B. pinni-caudatus predictably results in a steady loss of EODmasculinity and circadian rhythmicity over a week’s

Fig. 4 The amplitude and duration of EOD waveforms of B.pinnicaudatus increase at night. These day–night changes free-rununder both constant darkness and constant light, and thusconstitute true circadian rhythms. EODs are recorded automati-cally in the EOD machine (Stoddard et al. 2003). Repeatablerecordings of amplitude and waveform shape are obtained by

recording only when the fish is oriented lengthwise in an electricallytransparent shelter tube. s-p2 is the time constant of theexponential decay of second phase of the EOD, a parametricquality that tracks the duration of the second phase. The gray barsare periods of darkness

Fig. 5 Stressful social stimuliinduce rapid EODenhancement in both sexes oflab-reared B. pinnicaudatus.Shown here are typical datafrom a male (left) and female(right) forced to share a hidingtube during the day with a samesex conspecific (females respondsimilarly to intrusion by males).The EOD waveforms shownabove correspond to the timebefore the intrusion and thepeak of the response. Females’EOD enhancements are smallerthan males’ and are restricted tochanges in duration of theEOD’s second phase (measuredhere as s-p2), which increasespower in the low end of thefrequency spectrum (Stoddard2002)

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time. Restoration of a social environment, likewise,restores the masculinity of the EOD and its circadianrhythmicity. Restoration of EOD masculinity appearson two distinct timescales. Partial restoration occursover an hour or two, while full restoration is slower andtakes 2 days to 1 week, depending on the sexes and sizesof the social stimuli (Franchina et al. 2001). Thus weexpect to find two distinct biochemical processesinvolved in the regulation of the waveform, one fast-acting (e.g., minutes) and a second slow-acting (e.g.,days).

The more rapid masculinization of the EOD is pro-duced by peripheral action of melanocortin peptidehormones, including adrenocorticotropic hormone(ACTH) and a-melanocyte stimulating hormone(a-MSH). Intramuscular injection of either hormone invivo, or direct application to electrocytes in vitro, resultsin rapid increase in amplitude and second phase durationof the EOD of Brachyhypopomus on the same timescale(6–45 min) as observed during social interactions(Markham and Stoddard 2005; Fig. 6). Melanocortinpeptides are different cuts of the same pro-hormone,

produced by the intermediate lobe of the pituitary. Thesepeptides initiate their actions via five membrane-boundmelanocortin receptor subtypes (MC1R–MC5R) withdiverse roles and patterns of distribution (review Hadleyand Haskell-Luevano 1999).

The a-MSH analog SHU9119, which is a potentantagonist of mammalian MC3R and MC4R but anagonist at MC1R and MC5R (Hruby et al. 1995), mas-culinizes single-electrocyte potentials in a manner similarto ACTH (Fig. 6). Caution is in order when interpretingthe effects of ligands across taxa, but these results at leastsuggest that modulation of electrocyte APs is mediatedby activation of a receptor pharmacologically similar tothe mammalian MC1R or MC5R. Involvement of eitherisoform is plausible in the modulation of electrocyte APs.In addition to inducing cortisol release, another majorfunction of melanocortin action in the periphery isMC1R-mediated melanosome dispersal within melano-cytes as part of the stress-induced skin-darkeningresponse of fish and amphibians. The MC5R, widelydistributed in peripheral tissues and the primary isoformexpressed in skeletal muscle (Fathi et al. 1995), could be

Fig. 6 The EOD of B.pinnicaudatus is modulatedrapidly by serotonergic andmelanocortin agents. Solid linesrepresent baseline recordings.Dashed lines representrecordings 60 min afterpharmacological treatment.Intramuscular injections ofserotonin (5-HT) andmelanocortin peptides such asadrenocorticotropic hormone(ACTH) enhance bothamplitude and second phaseduration (s-p2) of the EOD inB. pinnicaudatus. Salineinjections performed quicklyand gently have no effect, andthe serotonin 1A receptoragonist 8-OH-DPAT reducesboth amplitude and s-p2 of theEOD. These effects areapparent in calibrated remoteEODs measured 50 cm fromthe fish and in EODs recordedby miniature electrodes at thetail surface. Additionally, themelanocortin ACTH enhancesamplitude and s-p2 of single-electrocyte potentials in vitro,whereas serotonin does not.The effect of ACTH on single-electrocyte potentials in vitro ismimicked by application ofSHU9119, a selective agonist ofthe mammalian melanocortinreceptor isoforms MC1R andMC5R. Bar charts presentmeans, with error barsindicating SEM

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retained during the developmental conversion of myo-cytes to electrocytes.

All melanocortin receptors are positively coupled toadenlyate cyclase, which often modifies cellular physi-ology through intracellular activation of protein kinaseA (PKA). Application of the membrane permeablecAMP analog 8-bromo-cAMP induces rapid enhance-ment of the EOD amplitude in vivo and in vitro inSternopygus and Brachyhypopomus, and prior blockageof PKA prevents these actions (McAnelly and Zakon1996; McAnelly et al. 2003; Markham and Stoddard2005).

In Sternopygus, the enhancement of EOD amplitudeis due to an increase in the magnitude of the Na+ currentwhich affects the EOD amplitude in two ways. First, thisincrease insures that each AP reaches the sodium equi-librium potential, ENa, so that each electrocyte generatesa maximum amount of current. Secondly, the increase inthe number of open Na+ channels during the AP de-creases the internal resistance of the electric organ. If theelectric organ is viewed as the battery of a Theveninequivalent circuit, and the resistances of this circuit aresimplified to the internal resistance of the electric organand the resistance of the water around the fish (Bennett1970), a drop in the internal resistance of the organ in-creases the voltage drop across the resistance of the water(McAnelly et al. 2003). In B. pinnicaudatus electrocytestreated with melanocortins or 8-bromo-cAMP, ampli-tude of the intracellular AP is almost invariant while theextracellular potential increases in amplitude. As before,extra current is passed outside the cell, increasing thevoltage drop across the media outside the cell. Unlike thecase in Sternopygus, however, the intracellular AP doesnot approach ENa (Markham and Stoddard 2005). Tomaintain a constant intracellular peak while increasingthe voltage outside the cell, the equivalent circuit mustrequire the resistance of the other excitable membraneface to decrease simultaneously with the inward Na+

current. Further work will reveal whether this model iscorrect.

Serotonin (5-hydroxytryptamine or 5-HT) appears tobe a significant central regulator of rapid EOD mascu-linity, with a time course close to that following melano-cortin injections. Intramuscular injection of serotonincauses a rapid enhancement of the EOD of B. pinnicaud-atus (Stoddard et al. 2003), but a direct application to theelectrocytes in vitro or the spinal cord in vivo has littleeffect (Markham and Stoddard 2003, 2005; Fig. 6). Weconclude its action lies upstream, probably in the pituitaryand/or hypothalamus. Unpublished pharmacology stud-ies have identified two distinct serotonin receptors in-volved in dynamic (�30–60 min) regulation of the EOD(Stoddard 2006). A receptor with the drug response pro-file of a 5HT2A receptor enhances the EOD, consistentwith the effects seen following 5HT injections. A secondreceptor,matching the profile of a 5HT1A receptor, exertsrapid control over EOD masculinity, but the response isinverted: antagonists enhance the EOD over an hour’stime and agonists cause EOD masculinity to wane

(Fig. 6). Interestingly, activation of the 5HT1A system istypical of subordinate individuals of many taxa. In sal-monids, for instance, 5HT1A activation darkens the skinof individuals on dark backgrounds, making them visu-ally cryptic (Hoglund et al. 2002a, b). Similarly, 5HT1Aagonists in B. pinnicaudatus reduce EOD amplitude andduration, making the individual electrically cryptic.Whatappears different is that 5HT1A activation in salmonidsenhancesmelanocortin release to darken the skin, whereasin the electric fishB. pinnicaudatus, it probably reduces themelanocortin release to diminish the EOD. This proposedinversion of the serotonin–melanocortin connection begsfurther investigation.

Long-term changes in the EOD

Changes in EOD waveform lasting days to months maybe activated by environmental stimuli that initiatebreeding, such as increased food availability, decreasedwater conductivity, or social stimuli, such as the intro-duction of a mate or a competitor into a tank. We do notyet know how these stimuli are ‘‘transduced’’ by theanimals, but these stimuli are known to regulate thelevels of various hormones, such as androgens orglucocorticoids, which in turn initiate long-term effectsvia their actions on steroid receptors.

Beginning with the work of Meyer (1983) numerousstudies have demonstrated the masculinizing effects ofandrogens on the EOD waveforms of various electricfish. 5a-dihydrotestosterone (5a-DHT) has been a valu-able tool in elucidating responses to androgens, becauseit binds androgen receptors and it cannot be aromatizedto estradiol. Slow-release implants of 5a-DHT in adultfemale B. pinnicaudatus masculinize the response of theEOD waveform to social challenges, serotonin, andACTH. These implants also masculinize (increase) cir-cadian rhythms in EOD duration, but fail to masculinizethe circadian rhythm in EOD amplitude (S. Allee et al.,unpublished data; Fig. 7). Together, these results sug-gest a strong peripheral effect of androgens on regula-tion of the EOD, though the incomplete masculinizationof female circadian rhythms in EOD amplitude couldresult from the maturity of the subjects or perhaps implyadditional contributing factors.

5a-DHT implants in adult Sternopygus lower EODfrequency, presumably by acting on the firing frequencyof the neurons in the pacemaker nucleus, and lengthenthe EOD pulse by broadening the electrocyte AP (Fewand Zakon 2001). The variation in EOD pulse durationof Sternopygus measured in the water outside the animalis reflected in differences in duration of APs measuredintracellularly: long duration APs in fish with low EODfrequencies and short duration APs in fish with highEOD frequencies (Fig. 2). Androgen treatment broadensthe AP much as it does the whole EOD pulse recorded invivo (Mills and Zakon 1991). The Sternopygus electro-cyte has three ionic currents involved in generation of theAP: an inward rectifier that sets resting potential, a Na+

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current (INa) that generates the rising phase of the APand whose inactivation initiates the falling phase of theAP, and an outward rectifier (IK) that repolarizes the AP(Ferrari and Zakon 1993). In this system INa and IK areuniquely co-regulated with great precision to generatethe fourfold variation in AP duration seen in Sternopy-gus. Fish with long APs have slowly inactivating INa andslowly activating IK, thereby broadening the AP, whilefish with short APs have rapidly inactivating INa andrapidly activating IK, producing a shorter AP (Ferrariet al. 1995; McAnelly and Zakon 2000; Fig. 8).

The Zakon lab has cloned Na+ and K+ channelgenes from the Sternopygus EO in order to understandhow these currents are regulated. One major family ofvoltage-dependent K+ channels, the shaker or Kv1family, has eight members in tetrapods, and at least adozen in teleost fish (H. Zakon et al., unpublished data).Kv1 subunits are often associated with a delayed recti-fying current such as we observed in the electrocytes.Three different Kv1 genes are expressed in the Sterno-pygus EO: Kv1.1a, Kv1.2a, and Kv1.2b (P. Few and H.Zakon, unpublished data.). Kv1.2b does not show anyvariation in expression across the EOD frequency range,whereas Kv1.1a and Kv1.2a are expressed in high levelsin fish with high EOD frequencies, low levels in fish withlow EOD frequencies, and intermediate levels in fishwith intermediate EOD frequencies. Furthermore, if fe-males (who have high EOD frequencies) are treated with5a-DHT, Kv1.1a and Kv1.2a are suppressed, butKv1.2b levels are unaffected (Fig. 9).

Potassium channels form as a tetramer, and differentsubunits from the same channel family may co-associatewithin a tetramer. Let us assume that the levels of pro-tein of each subunit reflect the levels of its RNA and thatthere is no preferential association for particular pairs ofsubunits. Then we imagine that the K+ channel tetramerin fish with high EOD frequencies is formed primarily ofKv1.1a and/or Kv1.2a and that these subunits have ra-pid inactivation kinetics. Conversely, K+ channels fromfish with low EOD frequencies are formed primarily bytetramers composed of Kv1.2b subunits and have slowactivation kinetics. Finally, K+ channels from fish with

intermediate EOD frequencies have Kv1 tetramers witha mix of all the three subunits (Fig. 9).

These genes have not yet been studied in an expres-sion system. However, pharmacological dissections ofthe K+ current in the native environment of the elec-trocyte are largely in agreement with the above scenario.The electrocyte K+ current from fish with high EODfrequencies activates most rapidly and is sensitive to theK+ channel blocker tetraethylammonium (TEA); theK+ current from fish with low EOD frequencies acti-vates slowly and is less sensitive to TEA, whereas K+

Fig. 7 Sexually mature femaleB. pinnicaudatus develop male-like circadian rhythms in EODduration (s-p2) when implantedwith the non-aromatizableandrogen 5a-dihydrotestosterone(5a-DHT). Further, the 5a-DHTimplants augment the response tothe injections of the melanocortinACTH. EOD waveforms in thisfigure are normalized to themagnitude of the first EOD phase

Fig. 8 Kinetics of Na+ current inactivation and K+ currentactivation vary with EOD frequency and sex in Sternopygus. Toptraces are Na+ and K+ currents recorded under voltage clampfrom the electrocytes of a fish with a high EOD frequency (female).Bottom traces are these currents recorded from a fish with a lowEOD frequency (male). Note that the rates at which the Na+

current inactivates and the K+ current activates are faster infemales than in males (McAnelly and Zakon 2000)

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currents from fish with intermediate EOD frequenciesare intermediate in both properties (L. McAnelly and H.Zakon, unpublished data). In mammals, Kv1 homomersare highly TEA sensitive, Kv1.2 channels are far less so,and Kv1.1/1.2 heteromers are intermediate in theirsensitivity.

Sodium channels may be comprised of multiple su-bunits, but the conducting a-subunit is one large protein,which is a fully functional channel on its own. Thus,there is no way for two a-subunit gene products tointeract. All the voltage-gated Na+ channel genes havebeen cloned from Sternopygus and eight genes have beenfound (Lopreato et al. 2001; A. Novak et al., unpub-lished data). Two of these genes are orthologs of themammalian gene Nav1.4, a gene expressed only inmuscle in mammals. The Sternopygus genes, initiallynamed sterNa1 and sterNa6, are now named Nav1.4aand Nav1.4b, respectively. Nav1.4a is expressed in themuscle and the EO, whereas Nav1.4b is expressed onlyin the EO. In the EO, levels of Nav1.4b do not varyacross the EOD frequency, but those of Nav1.4a arehigher in fish with a high EOD frequency (Fig. 10).When fish are treated with 5a-DHT, levels of Nav1.4bare unaffected whereas levels of Nav1.4a are suppressed(Liu et al. 2005).

These genes are now being studied in an expressionsystem so that their biophysical properties will soon beknown. Given that Nav1.4a is in muscle, which pre-sumably makes a brief AP, and is abundant in the EO ofhigh-frequency female fish, which makes a short AP, wepredict that Nav1.4a generates a Na+ current thatinactivates ‘‘normally.’’ Nav1.4b, on the other hand, isexpressed only in the EO and shows many amino acidsubstitutions suggesting that it has undergone significantevolution for a role in social signaling (H. Zakon et al.,unpublished data). We suggest that it is slowly inacti-vating, but this must be verified by expression studies.

While the differential expression of these two genesmight, at the first glance, seem sufficient to explainvariation in Na+ currents, that is not the case based ontheoretical grounds. If Na+ currents are the result ofcurrent flowing through two different channels, then thekinetics of the inactivation of the currents will be best fitby a bi-exponential decay time constant, each exponentreflecting the exponential decay of each current. Themagnitudes of each exponential term rather than therate of decay should vary with EOD frequency. How-ever, what is observed is that the inactivation timeconstant is well fit by a single decay function whose timeconstant varies across the EOD frequency range.

Ion channels have accessory proteins that are involvedin either targeting the channels to the membrane ormodifying the channels’ properties. Sodium channels inmammals have four different accessory proteins, called bsubunits, and one of these, b1, is expressed in muscle(Isom 2001). The addition of the b1 subunit increases therate of Na+ current inactivation when the b1 subunit isco-expressed with cRNA for mammalian Na+ channel infrog oocytes. Electric fish have a b1 subunit and it is ex-pressed inbrain andmuscle aswell as in electric organ (Liuand Zakon 2004). Since the b1 subunit speeds inactiva-tion, we would predict that it is more abundant in theelectrocytes of fish with high EOD frequencies. That is, infact, the case. However, the situation is further compli-cated by the fact that the b1 subunit is alternatively splicedin teleosts—a similar b1 subunit found in zebrafish isspliced identically (Hobson and Isom 2003). One of thesplice forms, the ‘‘short form,’’ is in greater abundance inthe EO of fish with high EOD frequencies, whereas the‘‘long’’ form does not vary with EOD frequency (Liu et al.2005). To date, only Nav1.4a has been expressed inXenopus oocytes. As expected, it generates a fast Na+

Fig. 9 Schematic diagram of the relationship between Kv1 geneexpression and EOD frequency in Sternopygus. ‘‘Fast’’ and ‘‘slow’’refer to the presumed rates of activation of each subunit in ahomomer. The colored doughnuts on the right illustrate hypothet-ical subunit composition of the electrocyte Kv1 channel of fish withhigh-, mid- and low-frequency EODs in accordance with therelative abundance of the mRNAs of each subunit in fish across theEOD frequency range (P. Few and H. Zakon, unpublished data)

Fig. 10 Schematic diagram of the relationship between Na+

channel gene expression and EOD frequency in Sternopygus.Nav1.4a and the short isoform of the b1 subunit are expressed inhigher levels in fish with high EOD frequencies and short EODpulses. We do not yet know how the two splice forms of the b1subunit associate with the two Na+ channels (Liu et al. 2005)

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current, and co-expression of Nav1.4a with either theshort or the long form of the b1 subunit speeds up currentinactivation. It will be interesting to test whether Nav1.4binactivates more slowly on its own, and how this is influ-enced by the b1 subunits. Finally, it will be intriguing tolearn how the interactions of two Na+ channels and twosplice forms of the b1 subunit give rise to a Na+ currentwith a simple mono-exponentially decaying inactivation.

Biomedical implications of EOD modulation

Understanding the long-term and short-term mecha-nisms by which gymnotiform electric fish modulate theirEODs has a number of important biomedical implica-tions. Because changes in the EOD waveform result di-rectly from changes in the intrinsic excitability ofelectrocytes, these fish offer exemplary systems toinvestigate both long- and short-term modulations ofintrinsic membrane excitability. Such intrinsic plasticityof excitable cells has a widespread importance for bothnormal and pathological functioning in a range of sys-tems. As we have discussed here, electric fish offer amodel system wherein intrinsic excitability is modifiedthrough at least two distinct classes of mechanisms. Forlong-term EOD modulations, hormones control theremodeling of the excitable membrane by changing theion channel populations. These effects may also serve topotentiate the signaling pathways involved in short-termEOD modulation. In the case of short-term EODmodulation, membrane-bound receptors initiate intra-cellular cascades that target working ion channels. Inboth cases, these changes in intrinsic excitability areaccomplished while maintaining a homeostatic balancethat enables ongoing function of the electrolocation andelectrocommunication systems.

Together with the synaptic plasticity of neural net-works, intrinsic plasticity is critical for proper func-tioning in most neural systems. The importantrelationship between synaptic plasticity and intrinsicplasticity is especially evident in the neural networkscontrolling rhythmic motor systems underlying behav-iors such as respiration, locomotion, and sleep rhythms(Calabrese 1998). In other areas, experience-dependentintrinsic plasticity plays an essential role in learning andmemory (review Zhang and Linden 2003), and intrinsicplasticity of neurons within the prefrontal cortex haseven been identified as potentially contributing to drug-seeking during cocaine withdrawal (Dong et al. 2005).Further investigations of the rapid and reversible mod-ulation of electrocyte excitability, as well as theremarkable co-regulation of multiple ionic currents toachieve a precise state of membrane excitability willlikely lead to additional insights on mechanisms ofintrinsic plasticity.

Inherited pathologies of intrinsic neural excitabilityare clearly associated with many forms of epilepsy (e.g.,Lossin et al. 2002, 2003), and similar disorders ofintrinsic excitability in peripheral tissues lead to disease

states in cardiac and skeletal muscle, and endocrinesystems (Ashcroft 2000). Gymnotiform electric fish haveevolved mechanisms for changing the performance ofhomologous channels to alter the meaning of the resul-tant communication signals. In so doing, these fish use arange of mechanisms to effectively transform ion chan-nels in the electric organ between physiological states.One possible approach to treating such disorders in fu-ture could be replacing pathological channels with nor-mal ones, but doing so presents the problem ofmaintaining functionality during such channel substitu-tions. The electric fish’s ability to remodel pathologicalchannels in situ suggests the possibility of alternativetreatment tactics.

In addition to being strong model systems forstudying intrinsic plasticity, the circadian rhythms inEOD waveforms of electric fish present unique oppor-tunities for investigating the neuroendocrine systemsinvolved in circadian rhythms and the factors that maylead to disruption or resilience of these systems. Thenormal functioning of circadian systems is readily ob-served in an organism’s sleep/wake cycles, the timing ofmealtime hunger, and cycles of overall activity. Lessobvious examples include circadian fluctuations inphysiological parameters associated with the onset andseverity of many medical conditions, such as dermatitis(e.g., Gelfant et al. 1982; Pigatto et al. 1985), asthma(e.g., Smolensky et al. 1999; Burioka et al. 2000; Panzeret al. 2003), and cancer (Keith et al. 2001; Filipski et al.2002; Fu et al. 2002). Circadian rhythms also areresponsible for variations in the effectiveness of manymedications (Andreotti et al. 1997) such as local anes-thetics (e.g., Bruguerolle et al. 1991) and chemotherapyagents (e.g., Levi 2001). Perhaps most importantly, cir-cadian rhythms are observed in numerous immune sys-tem functions (revs. Haus and Smolensky 1999; Roberts2000). Disturbances in the circadian rhythms, at anylevel of organization, have serious consequences for ill-ness, health, and well-being.

While the pacemaker components of circadian sys-tems are relatively well understood, much less is knownabout regulation of the output pathways in which thecycles of oscillator activity are ultimately expressed ascircadian rhythms in specific physiological or behavioralprocesses (reviews Dunlap 1999; Hardin 2000; Alladaet al. 2001; Reppert and Weaver 2001). Understandingcircadian output pathways is of particular importance inlight of the mounting evidence that disruptions in cir-cadian rhythms often result not from problems with thepacemaker, but instead from modulation or attenuationin particular circadian output pathways. This is the casefor some circadian disturbances associated with normalaging (review Monk and Kupfer 2000) and almost allstress-related disturbances in circadian function (e.g.,Richter 1967; Meerlo et al. 1997, 2002).

The EOD of weakly electric fish is a strong modelsystem for exploring the neuroendocrine mechanisms ofmodulation in circadian output systems. The EODwaveform and the amplitude of its circadian rhythms

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can be altered by changing the social or physical envi-ronment in an aquarium and can be induced chemicallyby injecting certain neuroactive compounds. Because theEOD is itself a socially significant behavior, changes inEOD waveform and rhythmicity are not simply assaysof circadian rhythmicity; these changes directly impactthe organism’s interaction with and fitness within itssocial environment. Thus, this system allows us toinvestigate the significance and mechanisms of modula-tion in the circadian EOD rhythms at all levels ofanalysis ranging from social interaction to neuroendo-crine events to ion currents in isolated membranes ofdissociated cells.

Acknowledgements The research was supported by NIH grantsMBRS GM08205 to PKS, NS025513 to HHZ, and MH064550 toMRM.

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