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Serotonin modulates the electric waveform of the gymnotiform electric fish Brachyhypopomus pinnicaudatus

Philip K. Stoddard^*, Michael R. Markham and Vielka L. Salazar

Department of Biological Sciences, Florida International University, Miami FL 33199, USA

View larger version (17K): [in a new window]   Fig. 1. Experimental tank used to record calibrated EODs of free-swimming fish. When the fish passed through, or rested inside the ceramic tube, electrodes on the tube informed the fish-in-tube detector of the fish's presence. Up to nine consecutive EODs were digitized from electrodes at the ends of the tanks while the fish-in-tube threshold criteria were met. ADC, analog-to-digital converter.

View larger version (11K): [in a new window]   Fig. 2. Parameters measured in each digitized EOD waveform. Amplitude is measured peak-to-peak using cubic spline interpolation to improve accuracy. The duration of each phase (dur) is calculated 10% of the distance from the zero-crossing to the peak. The time constant -P2 of the repolarization phase is estimated by fitting an inverse exponential to the middle 80% of the 2nd phase.

View larger version (29K): [in a new window]   Fig. 3. Offline data analysis using polynomial regression to remove the circadian rhythm from the pharmacological response. (A) The raw data are shown with a fourth order polynomial least-squares regression fitted to segments of data before the injection and after the treatment had worn off. The polynomial is subtracted from the data to produce the residual data (B). A second, higher order polynomial is fitted to the residual data. The peak response and 50% peak response points are calculated for this polynomial.

View larger version (21K): [in a new window]   Fig. 4. As a brief demonstration of the circadian rhythm, we left the lights on (A; male 029) or off (B; male 042) after completing an experiment. Under constant light or constant dark, EOD repolarization time (-P2) and amplitude (not shown) continue to oscillate on a shortened circadian rhythm, though the rise under free-running conditions is not as steep as occurs immediately after lights out in laboratory conditions. Vertical dashed lines indicate the previous times of lights on or off.

View larger version (18K): [in a new window]   Fig. 5. Effects of saline, serotonin, and male—male social interaction on the EOD waveform. (A) Effects on EOD amplitude and repolarization time (-P2). Changes due to circadian rhythm have been removed as described in Materials and methods (Fig. 3). (B) EOD waveforms sampled immediately before injection and at the time of peak effect. In the case of the saline injection (male 122) the two waveforms superimpose almost completely. Effects of serotonin varied between individuals. In some males (e.g. male 042), amplitude and -P2 both increased, whereas in others (e.g. male 036), repolarization time increased alone. Both males received saturating doses of 5-HT as indicated in Fig. 7 (male 042: 2.5 nmol g-1; male 036: 25 nmol g-1). Note the difference in the time courses of the -P2 response as well (see Fig. 8). As part of a different experiment, male 222 had a second male placed briefly in his hiding tube (first broken line). EOD amplitude and -P2 increased in a manner similar to EODs of fish injected with serotonin, then declined following the second male's removal (second broken line).

View larger version (16K): [in a new window]   Fig. 6. Percentage change (mean ± 1 S.D.) in responses of EOD waveform parameters to injection of serotonin at doses 2.5 nmol g-1, or to saline controls. EOD parameters were measured as in Fig. 2. Means were compared with two-sample t-tests; the P values shown are one-tailed. Compared to the controls, serotonin caused significant increases in amplitude, second phase duration (dur-P2), and second phase repolarization time (-P2), but not of first phase duration (dur-P1).

View larger version (15K): [in a new window]   Fig. 7. The serotonin dose—response curve for repolarization time (-P2) showing means (horizontal bars) and S.E.M. (vertical bars) for each dose, and a least-squares fit logistic function for all data. The response saturated at 2.5 µmol l-1 g-1, the dose we used for further experiments. The mean serotonin-induced rise at saturating doses is approx. 2/3 that of the circadian rhythm. The dose—response curve for amplitude was not significant and is not shown. Filled triangles, saline controls; open triangles, 5-HT-dosed.

View larger version (25K): [in a new window]   Fig. 8. Time courses of EOD duration and amplitude responses for 5-HT doses of 0.75 nmol g-1 and higher. Points plotted are pre-injection baseline, 50% of peak, peak response, and response half-life. (A) Dur-P1 (duration of the first EOD phase) showed no clear response to 5-HT. (B) -P2, (repolarization time of the second phase) showed the clearest response, with a bimodal distribution of peaks occurring at approx. 25 and 90 min after injection. (C) Amplitude was variable; if it rose, it peaked by approx. 30 min.

View larger version (17K): [in a new window]   Fig. 9. 5-HT induced rise in EOD repolarization time (-P2) versus rise in amplitude, expressed as a percentage of the day—night swing, illustrates how the serotonin-induced changes in repolarization time and amplitude are semi-independent. Note that most of the data fall above the 1:1 ratio (broken) line, indicating that both variables may change or repolarization time may change alone, but amplitude never changes without a change in repolarization time. Values shown are for males injected with 5-HT doses 0.75 nmol g-1 and higher.

View larger version (26K): [in a new window]   Fig. 10. (A) The response of repolarization time (-P2) (Ai) and amplitude (Aii) to 5-HT doses 2.5 nmol g-1 varies as a function of the magnitude of the circadian swing. Thus individuals with large circadian oscillations in -P2 or amplitude were predisposed to increase -P2 or amplitude more when injected with 5-HT than were fish with smaller cycles. (B) Daily minima and nightly maxima of -P2 (Bi) and amplitude (Bii) are tightly correlated. Regression (solid) lines are forced through zero, and the unity slope (broken) line is shown for comparison. The difference between the solid regression line and broken unity slope line represents the day—night swing. Thus, the larger the baselines of -P2 or amplitude, the larger the nightly swings and the larger the responses to 5-HT.