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First published online April 17, 2009
Journal of Experimental Biology 212, 1351-1364 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022566

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Waveform generation in the weakly electric fish Gymnotus coropinae (Hoedeman): the electric organ and the electric organ discharge

María E. Castelló, Alejo Rodríguez-Cattáneo, Pedro A. Aguilera, Leticia Iribarne, Ana Carolina Pereira and Ángel A. Caputi^*

Departamento de Neurociencias Integrativas y Computacionales, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, CP 11600, Montevideo, Uruguay

View larger version (32K): [in this window] [in a new window]   Fig. 1. Gross anatomy of the electric organ as observed in frontal sections of the fish body. (A) Photograph of a specimen of G. coropinae in which the vertical lines (a to g) indicate positions of sections shown in the diagrams in B. (B) In each diagram some non-electrogenic structures are shown in different gray tones, and the tubes that contain the electrogenic tissue are shown in red. Three portions of the EO can be clearly distinguished: (1) subopercular, in which the EO consists of a medial and a lateral columns (section a); (2) abdominal, in which the EO is included in the abdominal wall (section b); and (3) main, along the rest of the body in which the EO occupies the region between the ventral muscles and the fin (sections c to g). A relatively large posterior electromotor nerve is present in the caudal region (in black in f and g) and a jelly-like material between the dorsal muscles, similar to that present between the electrocytes in the EO tubes (in green in e to g).

View larger version (120K): [in this window] [in a new window]   Fig. 2. Computer aided 3-D reconstruction of the subopercular portion of the EO based on serial parasagittal sections of the ventral half of the head impregnated according to the procedure of Cajal and De Castro. Ventral (A) and dorsal (B) views of a 3-D reconstruction of the ventral half of the head (cleared profile) to show the relative position of the subopercular portion of the EO. The position of the cleithrum is indicated in A and B by the dashed line. (C,D) Same views as in A and B with higher digital zoom to show the arrangement of electrocytes of the subopercular EO. At each side of the midline, two columns of electrocytes are evident. One medial, containing two rostro-caudally elongated electrocytes (dark and light brown), one elongated in the dorso-ventral axis (dark green), and a cuboidal electrocyte beneath it (dark blue). The axis of the lateral column of electrocytes follows an oblique course. It consists of seven ribbon-like electrocytes with their main dimension in the frontal plane (pink, violet, light green, cyan, yellow, blue and magenta).

View larger version (109K): [in this window] [in a new window]   Fig. 3. Detailed organization and innervation pattern of the subopercular electric organ. Magnified views of the 3-D reconstruction showed in Fig. 2. The right medial column is shown from the frontal (A) and medial (B) view. (C,D,E) Ventral, frontal and caudal views, respectively, of the right lateral column. In all cases the cleithrum and the connective sheet that extends between the cleithrum and the operculum, separating the head from the rest of the body, are also shown. The whitish coloring indicates the innervated region of the electrocytes' surface. Four electrocytes form the medial column, three rostrocaudally aligned (medial electrocytes 1, 2 and 3: ME-1, ME-2 and ME-3, respectively), and a fourth one (ME-4), beneath ME-3 in the same parasagittal plane. ME-1 and ME-2 are elongated in the rostral-caudal and ventral-dorsal axis, and flattened in the lateral-medial axis. ME-1 is innervated at the ventral border and ventral side of its lateral and medial faces, whereas ME-2 is doubly innervated (the rostral and caudal borders). ME-3, with a ribbon-like shape, is mostly surrounded by the cleithrum bone (A,B). It is innervated on its caudal-dorsal face (B). ME-4 is cuboidal and is innervated at its dorsal-caudal face (A,B). The lateral column runs along an almost horizontal plane, parallel to the skin surface, with its head intruding below the connective sheet. It consists of seven ribbon-like electrocytes (L1 to L7 in C). Most of LE-1 and LE-2 are inside the head, LE-3 and LE4 are partially on both sides, and LE-5–LE-7 are caudal to the connective sheet (C). LE-1 to LE-4 are shorter, run perpendicular to the midline inside four rostral compartments (two dorsal and two ventral; C and D), and are innervated on their caudal borders (C). LE-5, which is of intermediate length, is innervated on its caudal border (C). The medial parts of LE-6 and LE-7 run diagonally with respect to the midline and are innervated on their caudal border and part of their ventral surface (C). The lateral half of these electrocytes bends up and hence the electromotor end terminals innervate their lateral surfaces (D,E).

View larger version (96K): [in this window] [in a new window]   Fig. 4. Electrocyte innervation of the head portion of the EO. Examples of the electromotor end terminals contacting electrocytes of the medial (A) and lateral (B) columns as revealed by the silver impregnation technique. (A) Photomicrograph of a parasagittal section of the rostral and dorsal part of the medial column containing ECM1. Note the thin connective sheet that delimits the column (black arrow) where the whole of ECM1 and the rostral end of ECM2 are present. These electrocytes are separated by a gelatinous tissue traversed by a bunch of electromotor axons that reach and innervate the caudal face of ECM1. Some fibers (white arrow) course caudally to reach the rostral face of ECM2. Note the proximity of ECM1 to the cleithrum (dotted outline). (B) Collage of microphotographs of successive optical section in horizontal planes of an electrocyte of the lateral column and its innervating electromotor fibers reaching its caudal border where they ramify profusely.

View larger version (100K): [in this window] [in a new window]   Fig. 5. The abdominal portion of the EO. (A) Microphotograph of a horizontal section of a Cajal and De Castro preparation showing its general organization: two columns on each side of the midline. The electrocytes of the medial and lateral columns are cuboidal (red asterisks) and fusiform (blue asterisks), respectively. The medial electrocytes are caudally innervated (red arrows) whereas lateral ones are innervated in their medial face and poles. Electrocytes occupy the medial region of compartments delimited by thin connective sheaths and filled with a gelatinous connective tissue. The black arrowheads indicate the septa between neighboring compartments of a medial column. (B) Computer 3-D reconstruction of the abdominal portion of the EO from serial horizontal sections as in A. Electrocytes are color-coded according to their type. Cuboidal electrocytes, aligned along medial columns, are innervated on their caudal faces (yellow). The lateral columns contain large fusiform electrocytes innervated on their medial faces and poles (blue). (C,D) Higher resolution micrographs show the innervation pattern of medial electrocytes in horizontal sections tangential to the surface (C) or at the middle (D).

View larger version (148K): [in this window] [in a new window]   Fig. 6. The main portion of the EO. (A) Microphotograph of a frontal section of a Cajal and De Castro preparation. Note that the dorsal column (1) is elongated in the medial-lateral direction. It extends laterally between the ventral muscle mass and the fin muscles, partially overlapping the others at the medial border. Columns 2–4 lie on a parasagittal plane. In this section only on column 4 is an electrocyte visible (asterisk), whereas in some of the others, electromotor axons run within the jelly between electrocytes (arrowheads). A thick electromotor nerve (black arrow) runs along the sagittal plane. The white dashed line indicates the parasagittal plane corresponding to the microphotographs shown in B–D, and the black dashed line indicates the parasagittal plane corresponding to the microphotograph in E. (B–D) Micrographs of a parasagittal section, showing the innervation pattern of a fusiform electrocyte of column 1. Note that the terminal branches of the electromotor nerve innervate the lateral electrocytes on their poles and on adjacent ventral surfaces. (E) Low magnification micrograph of a parasagittal section showing the three ventral columns and the spatial relationship between cuboidal electrocytes of neighboring columns 2–4. (F,G) A micrograph and a camera lucida drawing of two cuboidal electrocytes and their caudal innervation.

View larger version (15K): [in this window] [in a new window]   Fig. 7. The EOD waveform of G. coropinae depends on the recording conditions. The head to tail EOD (htEOD) of G. coropinae consists of six components (V1r, V3r, V2ct, V3ct, V4ct, V5ct). Classically the head to tail EOD has been used as a taxonomic tool for species identification. However, in the case of G. coropinae it shows important variations depending on the position of the recording electrode (near: black trace; far: blue trace) and also on load [compare waveforms obtained with the fish in the water (black trace) with the fish in the air (red trace)]. This indicates an important complexity at the earlier stages of the EOD. Recordings obtained with the air gap technique (red) show relatively larger and earlier wave components, in particular V3r. As is better appreciated in the magnified inset (bottom boxed region), htEOD recordings in water, obtained between two electrodes placed on the main axis of the fish at 4 cm (black) and 16 cm (blue) from the skin surface show how V3r is overcome by V2 as the distance increases.

View larger version (27K): [in this window] [in a new window]   Fig. 8. The spatio-temporal pattern of activity as evidenced by the multiple air gap procedure. (A) The voltage drop generated by different portions of the fish's body was simultaneously recorded with the fish isolated in air. For the sake of clarity, data from only three fish are shown. The rostral portion (a) corresponds to two-sevenths of the fish length, and the others (b to f) to one-seventh each. For each gap between electrodes the normalized regional waveforms were aligned with respect to the main positive peak of the head to tail recordings of each fish. Note the fast progression of the activation along the fish body (approx. 500 m s–1). The arrowhead in f indicates the neurally generated components preceding the activation of the tail region of the EO (one magnified example is shown as a gray trace). (B–E) Absolute amplitude of the waveform components of the EOD as a function of their origin along the EO (a to f as indicated in A, mean ± s.d., N=9). (B) V1r and V2ct; (C) V3r and V3ct; (D) V4ct (E) V5ct.

View larger version (63K): [in this window] [in a new window]   Fig. 9. Near field potentials along the rostral region of the fish body indicate the presence of a generator at the level of the operculum. (A) Color map showing the electric field perpendicular to the main axis of the fish body, recorded 2 mm away from the nearest point of the skin, as a function of the rostro-caudal position and time. The schema shows the rostral portion of the fish and exemplifies the flow of currents during V3r (black) and V3ct (gray). Color scale was non linearly adjusted [arctg (V)] to make the comparison between components easier. (B) Traces obtained at three different recording sites exemplify the inversion of V1r (blue) and V3r (red dotted line). (C–G) The amplitude of each of the components as a function of the recording position. There is a reversal point in the local field at about the limit of the head for V1r and V3r indicating a rostral source of these components (B–D). Conversely, the polarity of the other wave components did not change (B black dotted lines, E–G).

View larger version (21K): [in this window] [in a new window]   Fig. 10. Field potentials at two different times of the EOD. `Vector maps' were constructed by plotting the field vectors at the intersection of perpendicular and parasagittal lines, 5 mm apart, on the horizontal plane passing through the fish's main longitudinal axis. Raw data were interpolated to duplicate the number of vectors in each row and column. (A) `Vector map' of the field at the time of V3r and V2c (line labeled as A over the traces in C). Note that there is a single sink flanked by two sources (large gray shaded arrows). (B) `Vector map' of the field at the time of V3ct (line labeled as B over the traces in C). Note the larger distance between the sink and the source (large gray shaded arrow). (C) Selected recordings of field as a function of time taken near the fovea (red asterisk and trace) and lateral to the fish (green asterisk and trace) are compared with the head to tail EOD (blue trace).

View larger version (19K): [in this window] [in a new window]   Fig. 11. Characteristic curves of the fish's body equivalent sources. (A) The normalized htEOD waveform obtained in the single air gap with a loading resistance of 2 M (blue trace) and 15 k (red trace) varies with load, indicating a non linear voltage–current relationship of some wave components. In the case of both V3r and V3ct (B and C) data are well fitted by lines (V3r: r2=0.978, P<0.0001, N=439; V3ct: r2=0.99, P<0.0001, N=439). The ordinate crossing value is a good index of electromotive force whereas the slope is an index of internal resistance. Note the difference in slope (V3r: –0.085±0.001; V3ct: –0.07±0.007) suggesting a drop in resistance caused by the activation of the main portion of the EO. In the case of both V4 and V5 (D and E), the relationship departs from linearity, suggesting a load-dependent recruitment of the electrogenic membranes. This is compatible with the increase of the ratios V4/V3 (F) and V5/V4 (G) as a function of the currents generating the preceding peak.

View larger version (8K): [in this window] [in a new window]   Fig. 12. Co-evolution of neurally and peripherally determined mechanisms of EO activation. Peripherally determined intervals (between V3 and V4) and neurally determined intervals (between V2 and V3) were measured for the signals obtained from gaps b to e defined in Fig. 8 in three species and represented in a Cartesian plot. Each symbol corresponds to a gap and each color to a species (mean ± s.d.). Data from A.R.-C. MSc thesis work (unpublished). G. carapo (N=5) and G. omari (N=4). Data from each species are grouped in a well-separated cluster from the other species.

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