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First published online February 12, 2007
Journal of Experimental Biology 210, 800-814 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.000638

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Post-natal development of the electromotor system in a pulse gymnotid electric fish

Ana Carolina Pereira¹, Alejo Rodríguez-Cattaneo¹, María E. Castelló^1,2 and Angel A. Caputi^1,*

¹ Departmento de Neurociencias Integrativas y Computacionales, Instituto de Investigaciones Biológicas Clemente Estable, Unidad Asociada de la Facultad de Ciencias, Montevideo, Uruguay
² Departmento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

View larger version (118K): [in this window] [in a new window]   Fig. 1. The electric organ of larval fish. Photomicrographs of coronal sections of 12 mm-long fish impregnated with silver at the abdominal region (A) and the middle region (B) of the fish body. As shown in A, in the abdominal region, the electric organ consists of pairs of tubes on each side of the body midline. A nerve is also evident, cut in cross section, lying at the dorsal edge between the lateral and medial electrocytes on each side (arrows). Thin branches innervating the caudal faces of the electrocytes emerge from these nerves (not shown here), indicating that they correspond to the anterior electromotor nerves observed in the adult. After the emergence of the anal fin, the electric organ consists of two sets of four tubes on each side of the midline (B). Note the dorsal–ventral decrease in the electrocytes' cross-sectional area. (C) Microphotograph of a semi-thin section of a 12 mm-long fish showing parts of two electrocytes. The striated appearance of the cytoplasm of one of them indicates its myogenic origin (black and white arrows point to the clear and dense phases of the stria, respectively). The periodicity of this striation is similar to that of skeletal muscle (inset), although it is much fainter.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Development of the electric organ (EO) from larval to adult stages. (A) Area of the fish body occupied by the EO (expressed as a percentage of the cross-sectional area) as a function of the distance from the jaw (expressed as a percentage of the fish length). The area of the fish body occupied by the EO in the rostral half of the fish body is greater in larvae (30 mm) than in juveniles (65 mm), and the contrary holds for the caudal half of the fish body. (B) For all studied fish (28, 68 and 185 mm), the density of electrocytes (expressed as the number of electrocytes per 1% of fish body length) increases from the rostral to the tail regions.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Characteristics of the pacemaker frequency as a function of fish length. (A) Raster of the first-order intervals emitted by a resting fish of 37 mm length. Note the regularity of electric organ discharge (EOD) interval, interrupted by accelerations, followed by slower relaxations up to the basal EOD interval (arrows). (B) Histogram of the inter-EOD intervals represented in A. Note the skew of the histogram that results from the accelerations indicated by the arrows in A. (C) Box plot comparing the individual means of the EOD intervals exhibited by fish of different lengths. The first category, fish shorter than 30 mm, discharged their EO at a rate significantly lower than the others. (D) Box plot comparing the variance of the EOD intervals exhibited by fish of different lengths. The EOD was significantly more regular for the two categories above 60 mm. (E) First-order frequency polygon of the inter-EOD interval normalized to the mean interval obtained from 45 fish ranging from 12 to 226 mm, showing the constancy of the distribution.

View larger version (5K): [in this window] [in a new window]   Fig. 4. Electric organ discharge (EOD) rate as a function of water temperature comparing larvae and juveniles of different length, gathered in the same day. Note that in all specimens there is a linear relationship with similar slope and that the smaller fish have a lower rate.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Novelty responses elicited by electrosensory stimuli. (A,B) Mean ± s.d. (N=10) of the inter-EOD (electric organ discharge) interval normalized to basal EOD interval (upper dashed line=100%), plotted as a function of time. The arrows indicate the moment when two wires are short circuited in front of the fovea. (A) Biphasic larvae exhibit larger and longer-lasting accelerations than (B) juveniles and adults in response to changes in reafferent signals. (C) The amplitude of the novelty response (defined as the maximum shortening of the inter-EOD interval as a percentage of the basal interval) decreases with fish length. (D) The half relaxation time (defined as the time in which the interval returns to values equal to basal minus 50% of the amplitude of the response) also decreases with fish length.

View larger version (16K): [in this window] [in a new window]   Fig. 6. The electric field generated by the electric organ discharge (EOD). (A) Typical EOD waveforms corresponding to biphasic larval, triphasic larval and juvenile stages, showing the progressive incorporation of EOD components. (B) The root mean square value of the EOD associated field (rmsEOD) is a linear function of the square of the fish length. This relationship holds for larvae, juveniles and adults, in spite of the differences in EOD waveform. (C) During the larval and juvenile stages, the frequency at which the power spectrum of the EOD peaks, increases with fish length.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Development of the electromotive force of the fish's body. The equivalent source was characterized with the air gap procedure (Cox and Coates, 1938; Caputi et al., 1989) by maintaining the fish body out of the water and measuring the drop of voltage across a known resistor. (A) Waveforms obtained using load resistors of 100 M (red) and 1 k (black). All waves except V4 show a similar time course (note that waveforms are shown slightly shifted in time for the sake of clarity). (B) Characteristic curves of the equivalent electromotive force of the whole fish body for V3 in a larvae (28 mm), a juvenile (68 mm) and two adults of different length (100 and 225 mm). (C) The electromotive force at the peak of V3 plotted as a function of fish length. Two populations gathered in consecutive summers are compared. (D) As observed in A, V4 is modified by an external load on the electric source in the larva and the juvenile but not in the adult. We characterized this phenomenon by the increment of the ratio V4/V3 caused by changing the load from 1 M to 1 k. The plot in D shows that this parameter is negatively correlated with fish length (r2=0.005, N=20), suggesting that maturation is a factor involved in this phenomenon.

View larger version (22K): [in this window] [in a new window]   Fig. 8. The spatio-temporal pattern of the equivalent electromotive force along development was studied using the multiple air gap technique [a procedure that allowed the identification of the generation site of the different waveform components of the electric organ discharge (EOD) (Caputi et al., 1993)]. (A) The 30 mm fish exhibited a three-phase EOD with a slow early negative component generated at the abdominal region, a positive component generated all along the body, and a late negative component generated at the tail region. (B) The last component to appear (V2) was clearly identified in the fish of 47 mm length (second trace, arrowhead) and was completely developed in (C) the juvenile of 68 mm length. Note the progressive reduction of the EOD duration and the relative increase of the late negative component (V4).

View larger version (27K): [in this window] [in a new window]   Fig. 9. Regional contribution of the fish body to the electromotive force during development. The contribution of regions corresponding to 1% of the fish length was estimated by plotting the regional electromotive force normalized by the length of the fish region in which it is generated (electromotive force per 1% of fish length) as a function of the position of the center of the region along the fish body (distance from the jaw expressed as percentage of fish length). The early negativity shows two components of different spatial origin and maturation times. In larvae (A,B), the generated electromotive force is largest at the rostral portions of the generating electric organs and shows a slow decay as a function of the distance from the jaw. Note that the peak of the curve moves rostrally as the fish grows (arrows in B). This coincides with the smooth wave observed in time-based recordings and therefore should be classified as V1 (light gray box). (C) A second component appears in juveniles of about 47–52 mm length and (D) is clearly defined beyond 55 mm of fish length. This component corresponds to the fast component generated at the center of the fish body, V2 (dark gray box).

View larger version (15K): [in this window] [in a new window]   Fig. 10. Further evidence of the different mechanisms of generation of V1 and V2 can be obtained by fish curarization, which blocks transmission at the neuro-electrocyte junction. The dose was adjusted to be just enough to stop breathing but not enough to completely block the discharge. (A) The head-to-tail equivalent electromotive force of a fish in control (black) and curarized (red) conditions. (B) The spatial profile of the first negative deflection in control (black) and curarized (red) conditions. Note the differential effect on V1 and V2; V1 is the less affected component because it is generated by the subthreshold activation of the rostral membranes of doubly innervated electrocytes. V4 is absent because the amount of current generated by V3 is subthreshold for generating an action potential at the rostral faces of single innervated electrocytes.

View larger version (22K): [in this window] [in a new window]   Fig. 11. Development of (A) V3 and (B) V4. To compare the data from fish of different length (i–iv), we calculated an electrogenic index: the electromotive force divided by the length (expressed as a percentage of the fish length) of the generating region. In all fish, there is a head-to-tail progressive increment of the electrogenic index with percentage of fish length. Note that both the maximum amplitude and the slope of the curves of both waveform components increase as fish grow. The increase of the growth coefficient for V4 is larger than for V3.

View larger version (40K): [in this window] [in a new window]   Fig. 12. (A) Schematics of the electrogenic system in the adult fish [modified from Caputi (Caputi, 1999)]. The activation of the electric organs (EO) in the biphasic larvae (B), in the triphasic larvae (C) and in juveniles and adults (D) is indicated in a schematic based on the adult electrogenic system. Arrows of different colors indicate the different waveform components that are progressively added in the course of postnatal development.