The Journal of Experimental Biology 202, 1387Ð1398 (1999) 1387 Printed in Great Britain © The Company of Biologists Limited 1999 JEB2091

WAVEFORM DISCRIMINATION, PHASE SENSITIVITY AND JAMMING AVOIDANCE IN A WAVE-TYPE ELECTRIC FISH

BERND KRAMER* Zoological Institute, University of Regensburg, D-93040 Regensburg, Germany *e-mail: [email protected]

Accepted 15 February; published on WWW 21 April 1999

Summary The electric organ discharge (EOD) of most species of frequency-clamped and phase-locked to the EOD the freshwater knifefishes (Gymnotiformes) of South (frequency difference 0 Hz). Opening the electronic America is of the wave, not the pulse, type. Wave EODs are feedback loop immediately restored discrimination usually of constant frequency and amplitude, and show a performance on an on/off basis, and a strong jamming bewildering multitude of species-characteristic waveforms. avoidance response (JAR; a frequency shift away from the The EOD of Eigenmannia is sexually dimorphic in stimulus) accompanied every behavioural decision (to go waveform and in the intensity of its higher harmonics. In for a food reward). The strong habituation of the JAR that a go/no go paradigm, trained food-rewarded fish occurs in response to stimuli of no behavioural discriminated between these waveforms, and naive consequence (the usual test situation) was not seen in the (untrained) fish showed a significant preference. To present experiments. The proposed sensory model (which determine whether spectral or waveform (time) cues are is based on time-marking T electroreceptors) is supported used by the fish, artificial stimuli of identical amplitude by these experiments, and a biological function for the JAR spectrum were synthesized that differed only in phase Ð subserving EOD waveform discrimination is shown to be relationship between their harmonics, i.e. waveform, and useful in a social context. the fish discriminated even among these stimulus waveforms (i.e. spectral cues are not required). Our sensory model predicts that, for successful waveform Key words: electric organ discharge, Gymnotiformes, signal detection, a minimum frequency difference is required waveform discrimination, amplitude spectrum, phase spectrum, between the stimulus and the EOD. As expected, trained frequency difference, jamming avoidance response, beat analysis, fish confused test stimuli of different waveform that were sensory mechanism, Eigenmannia.

Introduction In rivers and lakes of tropical South America, gymnotiform them, so that they represent a continuous wave (like a sine fish lead a secretive, nocturnal life. During the day, they stay wave) and are of a constant frequency that may be modulated out of the way of visually oriented predators by hiding and when the fish is excited. Fed into a loudspeaker, wave EODs moving little; they do not communicate using sound, which sound like a musical instrument playing a constant tone. would be detected by predatory catfish, cichlids and piranhas. The African weakly electric fish, the Mormyriformes Knifefishes communicate using electric organ discharges (elephantfish, snoutfish), consisting of approximately 200 (EODs) they sense with electroreceptor organs distributed all freshwater species that are only distantly related to the over their skin (for reviews, see Hagedorn, 1986; Kramer, Gymnotiformes, are all of the pulse type except for a single 1990, 1994, 1996; Moller, 1995; for more detailed reviews on species that is of the wave type. This contrasts with South electric organs, see Bennett, 1971, Bass, 1986; on American freshwater electric fish (perhaps 120 species in all), electroreceptor organs, Zakon, 1986, 1988). A second function most of which are of the wave type (families Sternopygidae of the electric system of the knifefishes is electrolocation of and Apteronotidae sensu Mago-Leccia, 1994). The reason for objects (for reviews, see Bastian, 1990, 1994), which is not this difference between the two major groups of electric dealt with here. freshwater fish that occupy, as far as we know, the same There are two phenotypes of EOD: pulse and wave. Pulses general niche in their respective continents is unknown. are brief all-or-none events of the order of 1 ms or a few An advantage of the pulse phenotype in communication is milliseconds separated by much longer intervals. Fed into a its relative immunity to cross-talk by conspecifics: the loudspeaker, a buzzing sound or single clicks at very low statistical probability of EOD coincidences (causing signal discharge rates are heard. Wave EODs have no pauses between masking) is low because of a low duty cycle (the proportion of 1388 B. KRAMER the time that the signal is on to the time that it is off). Therefore, octave; Kaunzinger and Kramer, 1995) and is not matched by pulse fish follow a time-sharing strategy of communication, the technical filters. That is equivalent to stating that even very efficiency of which is often enhanced by certain forms of slow beats are detected and that the transition from the phase-locking of the EOD pulse to that of another fish (‘echo’ sensation ‘beat present’ to the sensation ‘beat not present’ is or preferred latency responses, etc.; for a review, see Kramer, an abrupt one (like a switch). 1990). Compared with pulse fish, wave fish come off badly Certain bats emit long echolocation calls of constant with respect to signal integrity because their wave EOD is frequency that are terminated by a brief period of frequency always ‘on’ and, therefore, superimposed on (or masked by) modulation (Griffin, 1958). The calls of these CF-FM-type bats that of nearby conspecifics. This is similar to what we mask the faint echoes that are of slightly higher frequency experience when trying to communicate in a noisy crowd of because of the Doppler shift when approaching a target (for people. reviews, see Neuweiler, 1993; Popper and Fay, 1995). To Therefore, we might expect wave knifefishes to be rather detect the echoes in spite of the very small frequency incompetent at electric communication (perhaps excelling in difference from the bats’ own calls, the bats must use very active electrolocation performance instead), but this is not sharp frequency filtering (up to 3500 dB per octave; Suga et borne out by the facts. The thresholds of wave fishes for al., 1976; Suga, 1990). Depending on how fast they approach frequency and intensity discrimination, a prerequisite for their target, these bats lower the tone frequency of their calls communication using ‘tone’ signals, are among the lowest for such that the tone frequency of the echoes is kept at the an octavolateral sense among all vertebrates (including the optimum filter frequency of their highly specialised auditory human). Within a certain frequency range, Eigenmannia, a system (Doppler compensation; Schnitzler, 1972). gymnotiform wave fish, discriminates a change of stimulus In contrast, the sensitive T electroreceptors of wave frequency corresponding to 1 % of a musical tone interval knifefishes are only weakly V-tuned (filter slope, only 20 dB (Kramer and Kaunzinger, 1991). The absolute threshold of per octave even on the ‘high’ side of the ‘best’, the EOD wave fishes for an external stimulus is similar to that of pulse frequency; for reviews, see Kramer, 1985; Zakon, 1988; fish (for a review, see Kramer, 1996). Fleishman, 1992), and these fish use a totally different strategy of signal detection and assessment, analysis of the temporal properties of beats. The temporal properties of beats and the Wave fish analyse beats sensory coding, central nervous and motor responses have been The underlying reason for this unexpected sensory acuity studied extensively by Bullock et al. (1972a,b), Scheich and became clear as a result of a paradoxical result: surgically Bullock (1974), Scheich (1977aÐc), Heiligenberg (1991) and abolishing the EOD of the wave fish Sternopygus macrurus others. However, exactly how these fish analyse beats has caused the electrosensory threshold of trained fish to rise steeply remained a matter of controversy to the present day. Our own (by approximately 30 dB; Fleishman et al., 1992). Clearly, this results on how frequency and intensity are detected fish did not profit from the weak stimulus being no longer (Kaunzinger and Kramer, 1996) are summarised in a sensory masked by its own strong EOD that had been abolished; on the model based on time-marking (T) electroreceptor afferences contrary, this fish experienced a serious sensitivity loss. (Kramer and Kaunzinger in Kramer, 1996; for a review, see Apparently, beating of the stimulus frequency against that of Kramer, 1999) and are not presented here. the EOD is required for the fish to detect a stimulus. This hypothesis was confirmed by a similar sensitivity loss when intact, electrically competent Sternopygus (Fleishman et Wave fish detect stimulus waveform al., 1992) and Eigenmannia (Kaunzinger and Kramer, 1995) Wave fish can afford to avoid sharp signal filtering because were stimulated at a frequency exactly equal to their EOD of their ‘intelligent’ use of beat analysis and they enjoy frequency or, especially for Eigenmannia, also one of the considerable advantages: in spite of detecting a wide range of higher harmonics of the EOD frequency. Such stimuli were stimulus frequencies, their electrosensory threshold and their detected only at greatly increased stimulus intensities. thresholds for frequency and intensity discrimination are Surgically abolishing the EOD of a wave fish or stimulating it exceedingly low (their electrosensory acuity is high). A cost at one of the harmonics of its EOD frequency (the fundamental (in the form of a sensitivity loss) of the wide working range of included) all have in common that there is no beating Ð the this system is apparent only for certain spectral lines, the EOD wave EOD and the stimulus mix according to their fundamental and its higher harmonics (a situation that, for instantaneous phase relationship (which was kept stable useful echoes, does not occur in CF-FM bats because of the throughout the experiment by an electronic device in the case Doppler frequency shift). Another advantage of the system of Eigenmannia; Kaunzinger and Kramer, 1995). However, used by these fish is an undistorted stimulus waveform, even the slightest frequency mismatch immediately restored because signal distortion is an unavoidable consequence of the sensory acuity of Eigenmannia, and the filter slope (a sharp filtering. Therefore, in addition to stimulus frequency and spectral line-reject filter, similar to a very sharp notch response intensity, wave fish could, in theory, also assess stimulus in an electronic filter) of this response is one of the steepest for waveform, which is the topic of the present article. a sensory filter in the animal kingdom (up to 5000 dB per Another requirement for stimulus waveform detection is a The EOD of a wave-type electric fish 1389 suitable communication channel. In contrast to sound and other sawtooth wave, of identical frequency and peak-to-peak mechanical forms of signal transmission, the waveform of a amplitude (or, alternatively, identical root mean square complex electrical signal remains unchanged with distance amplitude in case the ‘effective’ amplitude was the relevant from the signal source (except for a gradual deterioration of cue). Trained, food-rewarded fish had no difficulty with this the signal/noise ratio). The waveform of an electrical signal is task, nor with discriminating digitally synthesized (Kramer and also independent of the angle at which it is received, except Weymann, 1987) female from male EODs (Kramer and for a polarity reversal that occurs with certain positional or Zupanc, 1986). The play-back of female rather than male rotational changes of the sending with respect to the receiving EODs proved significantly more attractive to untrained fish of dipole. Therefore, the electrical communication channel does both sexes that were given neither reward nor punishment transmit potentially useful waveform information, and the (Kramer and Otto, 1988). electrosensory system of knifefishes may detect this From these observations, we concluded that Eigenmannia information because it does not discard that information by apparently discriminates any pair of waveforms that differ sharp sensory filtering. sufficiently in harmonic content, as seen in an amplitude When studying the gymnotiforms of the Amazon near spectrum. For example, the sine wave does not possess any Manaus, I was astonished by the multitude of different overtones, but the sawtooth wave is extremely rich in waveforms of EOD in wave-type fish (Kramer et al., 1981). overtones (which are integer multiples of the fundamental This diversity (Kramer, 1990, Figs 3.14Ð3.22) suggests that, in frequency). A similar difference, although much less addition to EOD frequency, which varies from approximately pronounced, exists for Eigenmannia EODs, female EODs 15 Hz to an exceptionally high 1800 Hz in different species, displaying overtones of weak intensity and male EODs EOD waveform variation is also species-characteristic and, displaying overtones of much stronger intensity (Fig. 1). therefore, must be the product of selection. Mate recognition Differences in overtone intensity are the physical basis for the by EOD frequency, as envisaged by Hopkins and Heiligenberg sensation of timbre or sound quality that enables us to (1978), seemed to be ineffective in our species-richer study recognize the various musical instruments or individual human area because of extensive overlap of EOD frequencies and the voices. wide range of species-characteristic frequencies; for example, To exclude signal discrimination by spectral analysis, or an 260Ð650 Hz in Eigenmannia virescens at 27 ¡C (Hopkins, electrosensory equivalent of timbre assessment, we used 1974). For so many wave species, as found near Manaus, the computer-generated waveforms of identical amplitude number of non-overlapping frequency ranges is insufficient in spectrum (hence, timbre) but different waveform. Two the available frequency space. In contrast, different species harmonically related sine waves, one octave apart in frequency display characteristically different EOD waveforms at a similar and of a weaker intensity by −3 dB for the higher harmonic, as frequency (e.g. Fig. 3.20 in Kramer, 1990), and even members is observed in a male Eigenmannia EOD, were added using of the same species, such as Eigenmannia lineata males and two different phase relationships: one in which the peaks of the females, may differ in EOD waveform (Fig. 1) (Kramer, 1985; two waves coincided (Fig. 2A, arbitrarily called ‘0 °’), and Eigenmannia lineata is considered a synonym of E. virecens; another with a relative delay of the higher-frequency wave by Planquette et al., 1996). one-quarter of a cycle, or 90 ¡ (Fig. 2E). This yielded two We tested whether Eigenmannia discriminates characteristically different waveforms of identical timbre; an electronically generated functions, such as the sine from the audio playback of these is indistinguishable to the human ear.

Fig. 1. Waveforms (A,B) of electric organ discharges in Eigenmannia, with A 0 C their associated Fourier amplitude spectra (C,D). The ordinates of the -10 left-hand diagrams are arbitrary linear -20 amplitudes (V), those of the right-hand 0 diagrams are amplitudes expressed as -30 dB attenuation relative to the strongest -40 spectral component of each waveform 0 1.6 3.2 4.8 6.4 0 1 2 3 4 5 (which is the first harmonic or fundamental in both cases). B 0 D

(A,C) Female; (B,D) male. Note the Amplitude (dB) -10 almost sinusoidal waveform of the Linear amplitude (V) female electric organ discharge -20 (EOD), the higher harmonics of which are of much weaker amplitude than in 0 -30 the male EOD, whose waveform -40 deviates markedly from a sinusoid 0 1.6 3.2 4.8 6.4 0 1 2 3 4 5 (Kramer and Otto, 1991). Time (ms) Frequency (kHz) 1390 B. KRAMER

A D

φ45 φ0 0 0

Fig. 2. Stimulus waveforms (AÐE), all of identical amplitude spectrum B E (F), as used in training experiments. φ90 Waveforms are composed of two φ11 Linear amplitude (V) 0 harmonically related sine waves (a 0 strong fundamental, f1, that is superimposed by its harmonic, f2, of Linear amplitude (V) twice that frequency and with an 02468 − amplitude of 3 dB re f1). AÐE differ C 0 Time (ms) only in the phase (φ) difference F φ between f1 and f2, where 0 -10 f f φ22 1 2 designates the temporal coincidence -20 of the peaks of the two sine waves 0 (arbitrarily called ‘0 ¡ phase -30 difference’) and φ90 designates a Amplitude (dB) -40 delay of a quarter of a cycle (90 ¡) 0 2468 0 1 2345 (Kramer and Teubl, 1993). Time (ms) Frequency (kHz)

Eigenmannia, however, clearly discriminated between these (thus forming a beat) is faithfully reflected by the firing rate of waveforms, again both in conditioned discrimination tests T receptor afferences (which is modulated back and forth in (Kramer and Otto, 1991; Kramer and Teubl, 1993) and when the case of beats, following the modulation of intervals observing spontaneous preferences in naive fish. In the between zero-crossings; Scheich, 1977aÐc). spontaneous preference tests, the more Eigenmannia-like 0 ¡ When an external EOD mixes with a fish’s own EOD, the signal was preferred over the 90 ¡ signal that resembles an adequate stimulus for T receptors is different for the left and Apteronotus albifrons EOD fairly well (A. albifrons is a right sides of the body (or head and tail region receptors, predator; some apteronotids prey on the tails of other depending on the relative positions of the two fish). The knifefishes; Lundberg et al., 1996). The two artificial test external EOD adds onto a fish’s own EOD for the receptors of waveforms need not be maximally different, as with the 0 ¡ and one body side (according to instantaneous amplitudes), 90 ¡ signal pair; a much smaller waveform difference, such as whereas for the receptors on the opposite side, the that between Fig. 2A and Fig. 2C, is sufficient for superposition is subtractive because these receptors face the discrimination (discrimination threshold, between 11 and 22 ¡ opposite polarity of the distant dipole generating the external phase shift of the second relative to the first harmonic; Kramer EOD (that is, the external EOD is perceived as inverted, or and Teubl, 1993). Therefore, spectral cues, or differences in 180 ¡ out of phase, by these receptors; see Fig. 9 in Kramer and timbre for a sound signal, are not required for Eigenmannia to Otto, 1991; Hopkins and Bass, 1981). Therefore, there are discriminate between two electrical stimuli of different slight time disparities between zero-crossings of the combined waveform. A purely temporal sensory mechanism was called signals for local T receptors on the two sides of the fish for. (Fig. 3). The magnitude of these disparities over time reflects The sensory mechanism of Fig. 3 is compatible with the the waveform of the external EOD at greatly reduced speed: above results in trained and naive fish, and the sensory one external EOD cycle (of the order of 0.0025 s) is physiology as established by Scheich, Bullock, Szabo and represented by one beat cycle (of the order of 0.25 s), the others (for a review, see Zakon, 1988). In this model, afferent duration of which the fish can control by adjusting its EOD responses from time-marking T electroreceptor organs, but not frequency (Kramer and Otto, 1991). A suitable central nervous from amplitude-coding P receptors, are the relevant ones. At system circuit for detecting temporal disparities between the high sensitivity and temporal precision, afferences from T afferent input of T receptors originating from different body electroreceptors respond to a fish’s own EOD with one action regions has been discovered (Carr et al., 1986a,b; Carr, 1990) potential per EOD cycle phase-locked to the zero-crossings of that builds on the fast electrosensory pathway of Szabo (1967). the EOD wave (it is not known whether to positive- or to Gymnotiform pulse fish also seem able to discriminate negative-going zero-crossings). Any spontaneous EOD between the slight variations among conspecific EOD frequency shift or frequency modulation caused by an external waveforms in an agonistic context (McGregor and Westby, EOD of different frequency mixing with a fish’s own EOD 1992), although a conditioned discrimination test has not yet The EOD of a wave-type electric fish 1391 been accomplished. Hopkins and Westby (1986) suggest ‘scan hypothesis can be tested. For example, in the sensory model of sampling’ as a possible sensory mechanism in which regular Fig. 3, fish should be unable to discriminate waveforms (at sequences of EOD superpositions in fish with a small least those of identical amplitude spectra) when the stimulus difference in discharge rate, separated by much longer sections and EOD frequency are identical (because no beats are present without superpositions, would be the relevant cue. If at frequency identity). This was tested in six fish that, in confirmed, such a slow mode of signal analysis would contrast preparation for the critical experiments, were trained to with signal analysis by trained mormyrids (Gnathonemus discriminate between two stimuli of identical amplitude petersii and Pollimyrus adspersus), which discriminate spectrum but markedly different waveform (Fig. 2A,E). These between low-rate playbacks of pulse EODs (conspecific, stimuli were played back in random sequence at a constant heterospecific or synthetic) immediately and reliably, after frequency difference of 50 Hz above the fish’s own EOD receiving only a few (or even only one) stimulus pulses in frequency (as determined immediately before stimulus onset). which no such sequences of superpositions occur (Graff and At such a large frequency difference, no jamming avoidance Kramer, 1992; Paintner, 1998; Paintner and Kramer, 1998). responses were evoked and, to avoid startle responses, a stimulus rise time of 500 ms was chosen. Modifying the experimental arrangement of our earlier Waveform detection depends on beat analysis studies, the six fish (14.6–16.5 cm total length) were trained to Wave fish such as Eigenmannia apparently require beats for go for a food reward 42 cm away from their daytime hiding discriminating between different stimulus waveforms, and this shelter (a porous pot; Fig. 4A) when the rewarded stimulus,

AB

00 Amplitude (V) Amplitude (V)

0 61218 24 30 0 6 12 18 24 30 Time (ms) Time (ms) 200 CD 200 100

100 s) µ 0 s)

µ 0 6 12 18 30 Time (ms)

0 Phase ( 0 12 18 30 -100

Phase ( Time (ms) -100 -200

-200

Fig. 3. The electric organ discharge (EOD) of a female Eigenmannia (400 Hz) superimposed on that of a conspecific in social context (A,B) and as probably analyzed by the T electroreceptor system (C,D). (A,C) The external EOD is that of another female; (B,D) the external EOD is that of a male (both of 30 % amplitude and at 450 Hz). (A,B) Additive superposition is shown as solid lines, subtractive superposition as dotted lines (representing the adequate stimuli for local T electroreceptors on the right and left sides of the body). One full beat cycle (20 ms) is shown centred. (C,D) Time disparities (or phase differences) between the zero-crossings of curves (A,B) as a function of time. Whether positive- (᭹) or negative-going (᭺) zero-crossings (A,B) are chosen is irrelevant because the time disparities between both curves represent the waveforms of the external EODs at greatly reduced speed; the only difference being a 180 ¡ phase shift relative to the beat cycle. At the large frequency difference between mixing EODs chosen here for illustration (50 Hz), the waveform resolution is rather crude; a more realistic frequency difference of 5 Hz (i.e. a beat cycle of 200 ms) yields a tenfold better resolution (Kramer and Otto, 1991). 1392 B. KRAMER

Fig. 4. (A) The test aquarium viewed from above (75 cm long, 40 cm wide, 45 cm A deep). R, pair of recording electrodes, in line with a porous pot (P), a swimming channel formed from plastic mesh (M) and R P R a feeder (F). T, glass tube that dispenses M F the food reward into the feeder. A1, A2, glass tubes that deliver ‘punishing’ air bubbles. S, pair of stimulation electrodes. A1 A2 Training and testing were carried out during daylight hours only, when the fish S stayed inside the shelter (P). A stimulus 70 delivered via electrodes S evoked the fish’s B 60 ‘go’ response. In training trials, a ‘go’ response to stimulus S+ was rewarded by 50 S+ food at F and to S− a ‘go’ response was SÐ 40 S+φ punished with air bubbles (at A1; A2 was SÐφ only used when the fish had habituated to 30 A1). In testing trials (as shown in B), Latency (s) 20 neither S+ nor S− was followed by reinforcement. The orthogonal electrode 10 arrangement (R, S) and the limits to the 0 fish’s swimming path (M) made possible 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 continuous recording of electric organ Trial number discharge (EOD) frequency even during electrical stimulation. (B) Discrimination between two stimulus waveforms in a trained Eigenmannia (test trials). Stimulus S+ is shown in Fig. 2A, stimulus S− in Fig. 2E. Trials 1Ð8, acquiring the association of S+ with a food reward, of S− with a light punishment, seen as dissociation of the response latency curves. Latencies greater than 60 s were truncated; both stimuli were +50 Hz re the fish’s EOD. From trial 9 onwards, two additional test stimuli were introduced into the randomized stimulus regime (S+φ, green; and S−φ, red; both stimuli were frequency-clamped and phase-locked with respect to the fish’s EOD but were otherwise identical to S+ and S−, respectively). Note that S+φ and S−φ evoked variable response latencies, indicating confusion of waveforms by the fish, while it continued to discriminate consistently between S+ and S−. This shows that, for discrimination between waveforms, a frequency difference between the fish’s EOD and the stimulus is required (this example shows fish 4, Table 1; R. Blasius and B. Kramer, unpublished results).

S+, was presented. They were supposed to stay inside their their own. Two test stimuli (S+ and S−) were identical with the shelter when the alternative stimulus, S−, was presented (for a two training stimuli, and the difference from the fish’s own period of up to 30 s). If necessary, fish were punished with a EOD frequency was also 50 Hz (30 Hz for fish 6; see Table 1). few air bubbles injected into their path when they responded In contrast, the other two test stimuli, S+φ and S−φ (see in spite of an S− being played. Using a stopwatch, the progress Table 1), consisted of presenting the same two waveforms at of learning was monitored as the time interval from stimulus the fish’s own EOD frequency (∆f=0 Hz). To achieve onset to a fish placing its mouth in the Petri dish that contained maintained, precise frequency identity even when the fish the reward. The observer was visually screened from the fish changed its own EOD frequency, the stimulus was frequency- by a sheet of cloth. clamped and phase-locked to the fish’s EOD (at a constant When response times for S+ and S− began to separate (those phase difference of 91 °; the definition of phase is based on for S+ shortening and those for S− lengthening), ‘test’ stimuli zero-crossings times; see Fig. 1 in Kaunzinger and Kramer, were included in the stimulus regimes (see below). The 1996). Therefore, a fish was unable to ‘escape’ from the randomized blocks design (Cochran and Cox, 1957) of stimulus with a frequency change, since the computerised stimulus sequence for training stimuli was expanded to electronic device (which was triggered cycle-by-cycle by accommodate a test stimulus every fifth stimulation while square-wave pulses derived from the fish’s EOD) followed its carrying on with the training stimuli (at an inter-trial interval EOD frequency without delay. [For a description of the basic of at least 1 min). Test stimuli (the duration of which was a device, see Kramer and Weymann (1987); a version expanded maximum of 60 s in case of no response) were followed by for phase-locking of sine-wave stimuli was used by neither reward nor punishment; it is exclusively the responses Kaunzinger and Kramer (1996); in the present article, a version to test stimuli that are reported in the present (and previous) capable of using ‘arbitrary’ waveforms that are phase-locked papers. was used. The word ‘arbitrary’ is popular with electronics Test stimuli were of four kinds, although only two stimulus companies at present advertising certain stimulus generators, waveforms, the same ones as for training stimuli, were used, although phase-locking is not offered.] and their sequence followed a randomized blocks design of By phase-locking the stimulus to the EOD at a selectable, The EOD of a wave-type electric fish 1393 dynamically constant phase difference, relative frequency drift (JAR) experiments (Watanabe and Takeda, 1963) had to be or jitter that might occur when using frequency-clamping followed precisely, with the additional requirement of without phase-locking, and hence undefined phase permitting the test fish to travel between the shelter and feeder relationships, were prevented (see Discussion in Kaunzinger (Fig. 4A). Therefore, the fish’s swimming path was strictly and Kramer, 1995). Further advantages were that the sensation limited to a straight line connecting its shelter with the feeder level (which depends on the phase difference of a stimulus of and the recording electrodes, by a narrow channel made of constant amplitude; Kaunzinger and Kramer, 1996) and the plastic mesh (maximum diameter 9 cm, which allowed the waveform of the mixed signal (composed of the EOD and the extremely flexible fish to turn around). Stimulus intensity was −1 added stimulus) were stable throughout the experiments. 156 µVpeak-to-peak cm at the fish’s head when facing the feeder Therefore, in these experiments, there were not even small inside its shelter. At that position, sensation level was 48 dB parts of a beat cycle present that might give a fish cues as to for a frequency-clamped stimulus close, but not identical, to the signal waveform with which it was being stimulated. the EOD frequency (based on thresholds determined by To record the EOD uncontaminated from the stimulus, the Kaunzinger and Kramer, 1995). For stimulation at frequency classical electrode geometry for jamming avoidance response identity and 91 ¡ phase difference between EOD and stimulus,

Table 1. Conditioned discrimination between two stimulus waveforms in Eigenmannia Median SIQ Sum of Mean of Dunn’s (s) (s) ranks ranks N comparison Fish 1 S+ 4 0.5 278.5 13.26 21 P<0.001 KW=66.29 SÐ 60 0 1554 74 21 P<0.0001 S+φ 10 2 885.5 42.17 21 P>0.05 SÐφ 9 2 852 40.57 21

Fish 2 S+ 8 1.5 204.5 12.03 17 P<0.001 KW=47.08 SÐ 60 0 986.5 58.03 17 P<0.0001 S+φ 19 9.5 594.5 34.97 17 P>0.05 SÐφ 18 12.5 560.5 32.97 17

Fish 3 S+ 9 3.5 432 18.78 23 P<0.001 KW=58.54 SÐ 60 0 1797 78.13 23 P<0.0001 S+φ 23 9.25 1013.5 44.07 23 P>0.05 SÐφ 21 8.25 1035.5 45.02 23

Fish 4 S+ 9 1 320.5 15.26 21 P<0.001 KW=47.19 SÐ 60 0 1174.5 55.93 21 P<0.0001 S+φ 60 0 1056 50.29 21 P>0.05 SÐφ 60 0 1019 48.52 21

Fish 5 S+ 7 1 287 15.11 19 P<0.0001 KW=46.92 SÐ 60 0 1204 63.37 19 P<0.0001 S+φ 9 7.5 725.5 38.18 19 P>0.05 SÐφ 10 4.25 709.5 37.34 19

Fish 6 S+ 6 7.25 535.5 28.18 19 P<0.0001 KW=64.25 SÐ 60 0 1890 99.47 19 P<0.0001 S+φ 11 12.75 933 49.1 19 P>0.05 SÐφ 8 11.25 827 43.53 19 S+ 10 10.75 831.5 43.76 19 zero P<0.01 SÐzero 58 46.5 1538 80.95 19

Median response latencies (s) differ significantly between stimuli (left-hand P values, based on KW, KruskalÐWallis ANOVA by ranks). SIQ, semi-interquartile range (s). S+, SÐ, test stimuli of constant frequency (with frequency difference re EOD); S+φ, SÐφ, frequency-clamped, phase-locked stimuli; S+zero, SÐzero, stimuli with identical frequency re EOD, not frequency-clamped. The fish were of Colombian origin, obtained through a direct-importing dealer. Zoologische Staatssammlung, Munich/Germany, accession nos: ZSM 28575 and ZSM 28576 (R. Blasius and B. Kramer, unpublished results). KW, KruskalÐWallis; N, number of tests. For stimulus waveforms, see Fig. 2A,E. 1394 B. KRAMER the sensation level was only 26 dB (based on thresholds performing a JAR (thereby restoring beats). This hypothesis determined by Kaunzinger and Kramer, 1996). was tested in fish 6 by introducing two more test stimuli (that Compared with our earlier experiments with unclamped test is, six stimuli that were all run in parallel, following a stimuli (Kramer and Otto, 1991; Kramer and Teubl, 1993), the randomised blocks design) which consisted of playing back the present experiments were more restrictive. For example, fish two signal waveforms (again of Fig. 2A,E) at a constant had to learn to swim through a narrow channel and for a longer frequency that was identical to this fish’s EOD frequency at distance to obtain a food reward. Stimulation across the centre stimulus onset (S+zero and S−zero). These stimuli were not of the swimming channel by widely spaced electrodes, as was frequency-clamped and, hence, were of constant frequency necessary here, yields a less natural field geometry and training throughout the experiment. situation than when a small dipole (a signal source that can be As expected, fish 6 discriminated between these new, localised) close to the feeder is used. unclamped test stimuli (P<0.01, Table 1) probably because, In spite of these constraints, all six fish learned to with the frequency clamp disabled, the fish quickly changed its discriminate the two test signals (of Fig. 2A,E), as shown by EOD frequency by an amount sufficient to restore its capacity the consistent difference in response latencies (an example is for beat analysis. This assumption is supported by the fish’s shown in Fig. 4B). As soon as a fish discriminated (P<0.01, confusion of the same stimulus waveforms when phase-locked MannÐWhitney U-test), the frequency-clamped, phase-locked to its EOD (S+φ and S−φ, as also observed in all other fish). test stimuli were introduced. Whereas response latencies to unclamped stimuli continued to be either short (S+) or long (S−), those for both frequency-clamped, phase-locked stimuli, The JAR is instrumental to waveform discrimination S+φ and S−φ, assumed all possible values between extremes, From the previous experiment, it follows that a fish that is often oscillating back and forth; that is, the fish were unable to stimulated at its EOD frequency must perform a JAR to discriminate (Fig. 4B). discriminate between different stimulus waveforms. This The hypothesis that the response latencies were independent hypothesis was tested in an additional five untrained of the test stimuli was rejected separately for each fish Eigenmannia (11.5Ð15.5 cm total length; Table 2, Fig. 5A,B). (P<0.0001, KruskalÐWallis one-way analysis of variance, In preparation for the critical tests, fish were trained to ANOVA by ranks; Table 1). As expected, response latencies discriminate between the same two waveforms as in the for an unclamped S+ proved significantly shorter than for an previous experiment (Fig. 2A,E). However, the frequency unclamped S− (P<0.01 in each fish; Dunn’s multiple difference from the fish’s EOD was smaller and varied between comparison test). In contrast, for the two phase-locked test 10 and 30 Hz, depending on the fish and at which frequency stimuli that were run in parallel (S+φ and S−φ), no significant difference learning progress seemed best. Discrimination differences emerged in any of the six fish (P>0.05). Even with between the two test stimuli, S+ and S−, was confirmed in each continued training, a significant difference is unlikely because, fish using the Mann–Whitney U-test (P<0.01). In combination for phase-locked stimuli, the differences between the sums of with previous reports (Kramer and Otto, 1991; Kramer and ranks (as determined for the KruskalÐWallis statistic) were Teubl, 1993; Table 1), the number of fish that have close to zero in all cases (Table 1). successfully performed the discrimination task is now 18 out Even though none of the fish discriminated between the of 18 tested by four experimenters. phase-locked test stimuli (S+φ and S−φ), individual differences The critical test of the above hypothesis began (1) when S+ were apparent. For example, fish 4 tended not to approach the and S− were replaced by S+zero and S−zero, respectively, and feeder when it was apparently uncertain about the exact nature (2) when S+φ and S−φ were added to the regime of test stimuli of the stimulus, and long latencies for both phase-locked (as defined for Table 1). The present test stimulus regime was stimuli occurred frequently. In contrast, fish 1 tended to adopt identical to that used for fish 6 of Table 1, except that, for the the opposite strategy by visiting the feeder in response to any five fish of Table 2, S+ and S− were omitted (four test stimuli −1 stimulus. Fish 1 also appeared to be more ‘resolute’ than fish rather than six). Stimulus intensity was 165 µVpeak-to-peak cm , 4 and sometimes disregarded punishment with air bubbles corresponding to a sensation level of 49 dB for a stimulus (even when boosted by a second device; Fig. 4A). The results close, but not identical, to the EOD frequency; at a phase for each of the six fish tested support the conclusion that fish difference of 35 ¡, the sensation level for frequency-clamped are unable to discriminate between the stimulus waveforms (of stimuli (S+φ and S−φ) was 29 dB. Training stimulus duration identical amplitude spectrum) when presented at maintained was up to 45 s, inter-trial interval was again at least 1 min and frequency identity with the EOD (∆f=0 Hz), in spite of an test stimulus duration again up to 60 s. Response latency was excellent discrimination between the same stimuli when the time from stimulus onset until the tip of the fish’s tail had presented at 50 or 30 Hz difference from the resting EOD left the shelter completely. frequency. These frequency differences were sufficiently large The results for the five new fish (Table 2) confirmed the that a JAR was not evoked. result (of fish 6, Table 1) that, even when the stimuli were However, a fish that is stimulated at its own EOD frequency identical in frequency with their own EOD, all fish with the electronic feedback loop disabled should be able to discriminated between the two test stimuli, S+zero and S−zero discriminate between the two waveforms, but only after (P<0.05 in each fish, Table 2). An initial frequency difference The EOD of a wave-type electric fish 1395 between the stimulus and the EOD, as used for fish 1–5 in an incorrect null hypothesis, H0), and discrimination was Table 1, is unnecessary and does not provide a cue for established at a significance level of only P<0.05 in this fish discrimination. In contrast, fish were unable to discriminate (Table 2). An F-test for fish 5 confirmed the result (that between the two phase-locked stimuli, S+φ and S−φ, when rejection of H0 is justified; F=7.408; Bonferroni’s P<0.01 for frequency identity with the EOD was maintained (P>0.05 in the comparison S+zero/S−zero, and P>0.05 for the comparison each fish, Table 2). S+φ/S−φ; normality test of data passed with P>0.10; differences After nine full repeats of test trials, fish 5 habituated to the between standard deviations not significant as shown by a punishment with air bubbles that was associated with the S−zero Bartlett’s test, P=0.1138). training stimulus and no longer appeared to discriminate On stimulation with S+zero, all fish responded by changing between stimuli, as measured by response latencies. This their EOD frequency (as measured by the frequency change habituation problem had been observed in other fish before but ∆R = actual EOD frequency minus resting EOD frequency, never as persistently as in fish 5, which finally ignored the determined immediately before stimulus onset). Except on punishment, visiting the feeder in response to any stimulus. three out of 71 occasions, these stimuli of ∆f=0 Hz evoked a Because using a more severe form of punishment was not frequency increase, confirming the results of earlier studies considered acceptable in these fragile fish, the data sampled (Kramer, 1987; Kaunzinger and Kramer, 1995). The time after the ninth repeat were discarded, and it was impossible to course of the frequency change (∆R, absolute values) evoked increase the sample size beyond nine in fish 5 using the present by S+zero was averaged in each fish, and individual differences technique. With such a small sample size, the nonparametric were revealed. Fish 1 showed the strongest responses: 5 s after KruskalÐWallis test is known to possess very little power (see, stimulus onset, its averaged ∆R was 7.86 Hz, whereas values for example, Siegel and Castellan, 1988; Motulsky, 1995), of 1.16 Hz, 1.85 Hz, 1.18 Hz and 0.75 Hz were observed in increasing the probability of a ‘type II’ error (failing to reject fishes 2–5, respectively (Fig. 5A).

Table 2. Conditioned discrimination between two stimulus waveforms in Eigenmannia Median SIQ Sum of Mean of Dunn’s (s) (s) ranks ranks N comparison Fish 1 S+ 10.1 4.65 191 12.73 15 zero P<0.001 KW=23.83 SÐzero 60 8.95 625.5 41.7 15 P<0.0001 S+φ 53.2 20.45 528 35.2 15 P>0.05 SÐφ 32.4 24.63 485.5 32.37 15

Fish 2 S+ 23.7 8.65 629.5 37.03 17 zero P<0.01 KW=42.81 SÐzero 60 2.75 1004.5 59.09 17 P<0.0001 S+φ 5.5 2.85 331.5 19.5 17 P>0.05 SÐφ 8.6 2.4 380.5 22.38 17

Fish 3 S+ 18.5 3.2 443 29.53 15 zero P<0.01 KW=33.29 SÐzero 43.1 8.13 762 50.8 15 P<0.0001 S+φ 7.3 3.43 219.5 14.63 15 P>0.05 SÐφ 15.4 7.4 405.5 27.03 15

Fish 4 S+ 12.7 7.63 445.5 29.7 15 zero P<0.001 KW=37.17 SÐzero 48.8 7.18 787 52.47 15 P<0.0001 S+φ 11 3.1 372.5 24.83 15 P>0.05 SÐφ 5.1 2.48 225 15 15

Fish 5 S+ 6.2 1.35 153 17 9 zero P<0.05 KW=14.24 SÐzero 35.4 8.7 267.5 29.72 9 P=0.0026 S+φ 4.4 0.75 123 13.67 9 P>0.05 SÐφ 9.0 3.75 122.5 13.61 9

Median response latencies (s) differ significantly between stimuli in each fish tested (left-hand P values, based on KW, KruskalÐWallis ANOVA by ranks). SIQ, semi-interquartile range (s). S+zero, SÐzero, stimuli with identical frequency re EOD, not frequency-clamped; S+φ, SÐφ, frequency-clamped, phase-locked stimuli. The fish were of Guyanan origin, obtained through a direct-importing dealer. Zoologische Staatssammlung, Munich/Germany, accession nos: ZSM 28567 - 28574 (M. Orth and B. Kramer, unpublished results). KW, KruskalÐWallis; N, number of tests. For stimulus waveforms, see Fig. 2A,E. 1396 B. KRAMER

B 9.5 A Eigenmannia 1 10 8 7.0 Eigenmannia 1 6 4.5 4 2.0 2 -0.5 0 0 1.1 2.2 3.3 4.4 5.5 6.6 7.7 0.2 1.3 2.4 3.5 4.6 0 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 9.9 11.0 6 4.0 5 Eigenmannia 2 Eigenmannia 2 3.0 4 2.0 3 1.0 2 0 1 -1.0 0 0 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 13.0 14.1 15.2 16.3 17.4 18.5 19.6 20.7 21.8 22.9

6 5.5 Eigenmannia 3 5 4.5 Eigenmannia 3 3.5 4 2.5 3 1.5 2 0.5 1 -0.5 0 0 1.1 2.2 3.3 4.4 0.7 1.8 2.9 4.0 5.1 6.2 7.3 8.4 9.5 Frequency change (Hz) 6.6 8.8 11.0 13.2 15.4 17.6 19.8 EOD frequency change (Hz) 6 3.5 Eigenmannia 4 5 Eigenmannia 4 2.5 4 3 1.5 2 0.5 1 -0.5 0 0 1.1 2.2 3.3 0 1.1 2.2 3.3 4.4 5.5 6.6 7.7 6.8 7.9 9.0 10.1 11.2 12.3 13.4 14.515.6 16.7Ê17.8

2.5 Eigenmannia 5 2.0 3.0 Eigenmannia 5 1.5 1.5 1.0 0 0.5 -1.5 0 1.5 2.6 3.7 4.8 5.9 7.0 0 1.1 2.2 3.3 4.4 5.5 6.6 1.5 3.3 5.1 6.8 8.6 10.3 12.1 Time (s) Time (s)

Fig. 5. (A) Averaged jamming avoidance responses of five trained Eigenmannia (see Table 2), as evoked by S+zero stimuli (of constant frequency and initial ∆f=0 Hz; maximum frequency error +0.08Hz with respect to the fish’s EOD). Ordinate, frequency change of electric organ discharge (EOD) with respect to EOD frequency at stimulus onset (the arrows show where the time is set back to zero). Each point represents the mean of 20 EOD cycles sampled within a time window of 220ms, using a 10 MHz clock. For each fish, between 9 and 17 individual curves (Table 2) were aligned such that the times of stimulus onset coincided, and averaged (values are means ± S.D.). (B) Averaged frequency change for the same five trained Eigenmannia, as evoked by S+zero stimuli. As in A, but individual frequency curves (N=9Ð17; Table 2) were aligned such that the times of ‘go’ for a food reward coincided (arrows), and averaged (M. Orth and B. Kramer, unpublished results).

Factors contributing to the high variability of the JAR are opposed to stimulus onset) coincided and were averaged as habituation, sex and age (Kramer, 1987). In the present before. The time of decision-making (to go for a food reward) context, a possible further factor was of interest: is the JAR was the moment when the fish left its shelter, i.e. when it required for successful waveform discrimination, as showed the ‘go’ response. determined by a conditioned behaviour? Therefore, the curves Associated with the ‘go’ response, an additional frequency were aligned such that the times of decision-making (as change superseding the one shown in Fig. 5A (where The EOD of a wave-type electric fish 1397 averaging is centred on stimulus onset) was revealed (Fig. 5B). V (ed. W. S. Hoar and D. J. Randall), pp. 347Ð491. London, New In all fish, a steep frequency increase was observed York: Academic Press. immediately preceding the decision to ‘go’; in three fish, ‘go’ Bullock, T. H., Hamstra, R. H. and Scheich, H. (1972a). The coincided with the steepest part of the frequency change. In the jamming avoidance response of high frequency electric fish. I. other two fish (fish 2 and 4) frequency continued to rise in a General features. J. Comp. Physiol. A 77, 1Ð22. gentler manner once the decision to ‘go’ had been taken. The Bullock, T. H., Hamstra, R. H. and Scheich, H. (1972b). The jamming avoidance response of high frequency electric fish. II. ‘go’-associated, marked frequency rise occurred within 3 s, and ∆ Quantitative aspects. J. Comp. Physiol. A 77, 23Ð48. the highest R values immediately followed ‘go’. In three fish, Carr, C. E. (1990). Neuroethology of electric fish. Principles of once they had reached the feeder, EOD frequency relaxed to coding and processing sensory information. BioScience 40, frequencies below or equal to their frequency before ‘going’. 259Ð267. Values of ∆R around the time of ‘go’ were very large given Carr, C. E., Heiligenberg, W. and Rose, G. J. (1986a). A time- that inter-trial intervals were usually only 1 min and that comparison circuit in the electric fish midbrain. I. Behavior and stimulus intensity was high. Short inter-trial intervals such as physiology. J. Neurosci. 6, 107Ð119. these, combined with the strong stimulus intensity applied Carr, C. E., Maler, L. and Taylor, B. (1986b). A time-comparison here, led to marked, long-lasting habituation in ‘classical’ JAR circuit in the electric fish midbrain. II. Functional morphology. J. experiments (Kramer, 1987). Habituation was not prominent Neurosci. 6, 1372Ð1383. in the present experiments: mean maximum ∆R values were a Cochran, W. G. and Cox, G. M. (1957). Experimental Designs, 2nd edition. New York: John Wiley. record 9.37 Hz in fish 1, and still remarkable in fishes 2–5 Fleishman, L. J. (1992). Communication in the weakly electric fish (3.87 Hz, 5.94 Hz, 2.95 Hz and 1.94 Hz, respectively). The Sternopygus macrurus. I. The neural basis of conspecific EOD ∆ greatest individual (non-averaged) R values were 14.13 Hz in detection. J. Comp. Physiol. A 170, 335Ð348. fish 1 and 11.07 Hz in fish 3. Fleishman, L. J., Zakon, H. H. and Lemon, W. C. (1992). Fish 5, which displayed the lowest ∆R values, was the one Communication in the weakly electric fish Sternopygus macrurus. that finally habituated completely to punishment with air II. Behavioral test of conspecific EOD detection ability. J. Comp. bubbles (see above), and the correlation does not seem Physiol. A 170, 349Ð356. accidental. The time course of the frequency change of fish 5 Graff, C. and Kramer, B. (1992). Trained weakly-electric fishes (very moderate ∆R at first, but a strong response at decision Pollimyrus isidori and Gnathonemus petersii (Mormyridae, making) clearly demonstrates that the JAR served a sensory Teleostei) discriminate between waveforms of electric pulse function in detecting stimulus waveform even in this fish. This discharges. Ethology 90, 279Ð292. Griffin, D. R. (1958). Listening in the Dark. Princeton: Yale suggests that fish 5 did discriminate between the two University Press. waveforms even after trial 9, when it stopped showing Hagedorn, M. (1986). The ecology, courtship and mating of differential ‘go’ responses. gymnotiform electric fish. In Electroreception (ed. T. H. Bullock One of the functions of the frequency change behaviour of and W. Heiligenberg), pp. 497Ð525. New York: John Wiley. Eigenmannia – be it called JAR or ‘active beat response’ (as Heiligenberg, W. (1991). Neural Nets in Electric Fish. Cambridge, suggested by Kramer, 1996) Ð is clearly to enhance the MA: MIT Press. detection of the EOD waveforms of other fish when discharge Hopkins, C. D. (1974). Electric communication: functions in the frequencies are too similar for beat analysis to be possible. social behavior of Eigenmannia virescens. Behavior 50, 270Ð305. Hopkins, C. D. and Bass, A. H. (1981). Temporal coding of species D. 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