J Comp Physiol A (1994) 174:633-642
Journal of
9 Springer-Verlag 1994
Waveform generation in Rhamphichthys rostratus (L.)
(Teleostei, Gymnotiformes)
The electric organ and its spatiotemporal activation pattern
A. Caputi, O. Macadar, O. Trujillo-Cen6z
Divisions of Comparative Neuroanatomy and Neurophysiology,Instituto de Investigaciones Biol6gicas "Clemente Estable",
Av. Italia 3318, CPl1600, Montevideo, Uruguay
Accepted: 11 October 1993
Abstract. Rhamphichthys rostratus (L.) emits brief pulses
(2 ms) repeated very regularly at 50 Hz. The electric organ shows a heterogeneous distribution of the electrocyte
tubes and the occurrence of three electrocyte types (caudally innervated, rostrally innervated and marginallycaudally innervated). In the sub-opercular region the
electric organ consists of a pair of tubes containing only
caudally innervated electrocytes. At the abdominal region the EO consists of three pairs of tubes. Each pair
contains one of the described electrocyte types. The number of electrocyte tubes increases toward the tail to reach
nine or ten pairs in the most caudal segments. In the
intermediate region most tubes contain doubly innervated electrocytes except the ventral pair that contains caudally innervated electrocytes. The caudal 25% contains
exclusively caudally innervated electrocytes. The electric
organ discharge consists of five wave components (V1 to
V5). Electrophysiological data are consistent with the hypothesis that V1 results from the activity of the rostral
faces of rostrally innervated electrocytes. V2 results from
the activities of rostral faces of marginally-caudally innervated electrocytes while V3 results from the activities
of caudal faces of most electrocytes. Curarization experiments demonstrated that V4 and V5 result from action
potential invasion and are not directly elicited by neural
activity.
Key words: Electrogeneration - Electric organ
cytes Electric fish Rhamphichthyidae
Electro-
Abbreviations: AEN1, anterior electromotor nerve 1; AEN2, anteri-
or electromotor nerve 2; BMB, boraxic methylene blue; CIE, caudally innervated electrocytes; EMF, electromotive force; EO, electric organ; EOD, electric organ discharge; I, current amplitude;
MCIE, marginally-caudally innervated electrocytes; MT, medial
tubes; PEN, posterior electromotor nerve; Ri, internal impedance;
RIE, rostrally innervated electrocytes;R1, load resistor; SAT, short
abdominal tubes; V, voltage amplitude
Correspondence to: A. Caputi
Introduction
The diverse group of South American electric fish (Order
Gymnotiformes) includes approximately 60 species usually separated in six families (Mago-Leccia 1978). These
fish are able to generate species-specific EODs differing
in amplitude, repetition rate and waveform. Adapted to
live in a variety of ecological niches they exhibit complex
behaviors that have been explored in detail only in a few
species (Hopkins 1983, 1988).
Most electrogenic systems show a common pattern of
organization: the EO consists of several tubes lying dorsal to the anal fin musculature and extending along the
fish body. With the exception of the Apteronotidae, in
which the adult EO is formed by modified spinal electromotor axons, in all other taxa the EO tubes contain
electrogenic units - electrocytes - of myogenic origin.
Variations involving both the morphology and the electrophysiological properties of the electrocytes have been
described in different species (Bennett 1971 ; Bass 1986).
Investigations dealing with the EOs and EODs of gymnotids have been mainly focused on species belonging to
five families (Sternopygidae, Hypopomidae, Apteronotidae, Gymnotidae and Electrophoridae), while
information concerning the remaining family (Rhamphichthyidae) is limited to behavioral aspects (Lissmann
and Schwassmann 1965; Bennett 1971; Schwassmann
1976; Bass 1986).
As stressed by Bullock (1984, 1986, 1993), proper
understanding of the evolution and functional organization of neural systems requires studies dealing with many
species covering the major branches of each taxonomic
group.
The present paper is concerned with the morphology
of the EO and the EOD characteristics of Rhamphichthys
rostratus (L.) (family Rhamphichthyidae). Technical
procedures developed during previous investigations on
Gymnotus carapo (Trujillo-Cen6z et al. 1984; Lorenzo et
al. 1988; Trujillo-Cen6z and Echag/ie 1989; Macadar et
al. 1989; Caputi et al. 1989; Caputi et al. 1993) have
provided the basis for this investigation.
634
A. Caputi et al.: The EO and EOD of R. rostratus
R. rostratus is a pulse fish with a multiphasic, highly
regular discharge. As previously found in G. carapo the
EOD waveform exhibits a spatio-temporal pattern determined by the EO structural organization.
Material and methods
Fish. Four large R. rostratus (54, 65, 68 and 70 cm) were captured
at night in the gate for migrating fish of Salto Grande hydroelectric
plant (Uruguay river, west limit of Uruguay state). This species has
been described for this biogeographical zone (von Ihering 1907;
Ellis 1913) and the taxonomic identification was made using Ellis'key. Taking into account that the taxonomy and phyletic relationships of the Gymnotiformes are under revision (Mago-Leccia 1978;
Fink and Fink 1981 ; Gayet and Meunier 1991) the species identification should be considered tentative. However, von Ihering (1907,
pp. 279 original in Portuguese) advanced that: "It seems very
probable that with abundant material from diverse regions, it will
be verified that the alleged species (R. rostratus rnarrnoratus;
R. rostratus reinhardti) lack support even as sub-species; in the
case to be grouped in a single species this must be named Rhamphichthys rostratus (L.)".
One of the collected specimens died during manipulations in the
gate. It was immediately fixed by immersion in 10% formaldehyde
and used for gross-anatomy studies. The other three animals were
kept alive in a large aquarium and used for both electrophysiological experimentation and finer anatomical analysis. Fish were treated
with special care to avoid injuries that might impair survival. After
obtaining the desired functional data, the animals were fixed by
perfusion as described in the following paragraphs. All morphological and functional findings have been referred to percentage of fish
length, with the tip of the head 0% and the tip of the tail 100%.
7%
b
15%
30%
Anatomicalprocedures. Deeply anesthetized fish (1 mg ethomidate/1
ml water) were perfused through the heart with 5% paraformaldehyde dissolved in 0.1 mol 91-1 phosphate buffer. Before passing
the fixative fluid, blood was removed from the vascular bed with a
fish-adapted saline solution. Both the washing solution and the
fixative were propelled with a peristaltic pump. Cross sections of the
fish body were cut at different rostro-caudal levels to explore the
distribution of the electrogenic tissue. For this purpose the scales
were removed and the vertebral column was cut with a fine saw;
soft tissues were sectioned with a very sharp knife. Each section (approximately 2 mm thick) was cleared with glycerol and
mounted between two pieces of flat glass. The more slender tail
portions were cut with a vibratome (in these cases sections were 400 gm
thick). Large sections were projected with the aid of a photographic
enlarger and the outlines of both the fish body and the EO were
drawn on paper sheets. The vibratome sections were observed under
a light microscope and the outlines of the relevant structures were
drawn with the aid of a camera lucida.
The different tubes composing the EO were dissected out and
immersed in buffered osmium tetroxide (1% OsO4 in 0.1 mol" 1-1
phosphate buffer). Osmicated tubes were cleared with glycerol and
studied under the microscope. In other instances the tubes were
disrupted and single electrocytes were isolated to be observed and
photographed under the microscope. Isolated electrocytes were also
dehydrated and epoxy-resin embedded. Semithin sections (1-2 gm
thick) were cut with a LKB Ultrotome and stained with BMB.
Series of ultrathin sections were mounted on Formvar-coated grids
and doubly stained with uranyl acetate and lead citrate. The fine
structure of the electrocytes was analyzed with a transmission electron microscope at different magnifications.
Electrophysiologieal procedures. The EOD was characterized using
the same methods applied to study G. earapo (Caputi et al. 1989,
1993). Briefly, the E M F and Ri of the whole fish body were cal-
50%
85%
GCh
~
25
mm
PEN
2 mm
9
H1
Fig. 1 a A schematized lateral view of the EO (stippled) of R. rostratus. The EO is represented as discontinuous and for the sake of
clarity natural proportions have not been maintained. Individual
electrocyte tubes are shown only in the sub-opercular and abdominal portions, while in the remainder intermediate and caudal portions the EO has been represented as a whole (the break shows
the point at which the individual tubes are no longer represented).
b Five transverse sections depicting the number of electrocyte tubes
at different percentages of the fish length (the tip of the head is
considered to be 0% and the tip of the tail 100%). The electrocyte
tubes appear stippled; in the drawings showing 7%, 15%, 30%
and 50% of the fish length, the arrows indicate the EO location.
The open areas correspond to the main muscular masses. Note
that only in the drawing representing a cross section at 85% of
the fish length are the lateral line nerves (LLN) and the posterior
electromotor nerves (PEN) shown. OC oral cavity; GCh gills
chamber; A C abdominal cavity
A. Caputi et al.: The EO and EOD of R. rostratus
635
Fig. 2. a Semischematic representation of the rostral
portion of R. rostratus (ventral view). The left side of
the picture shows the three
electrocyte tubes and the two
anterior electromotor nerves
(AEN1 and AEN2). The
medial tube (horizontal shading) contains only CIEs. The
more lateral tube (diagonal
shading) only contains RIEs,
while the intermediate tube
(stippled) contains MCIEs.
b Low power microphotograph showing two CIEs
contained in the medial tube.
The arrowheads indicate the
axon bundles arising from
the AEN1. The thick short
arrow points rostrally. • 40
culated measuring voltage drop and current across known Rls. For
this purpose fish were suspended in air and tap water was circulated
through the gills.
Two electrodes, one placed in the mouth and the other in the
tip of the tail, were connected by known Rls. V and I amplitudes
were measured on the oscilloscope screen. Since V was found to be
a linear function of I (V= E M F - R, 9 the ordinate intercept and
the slope of the line were considered as good estimators of the EMF
and the R~, respectively. The locally generated discharge was recorded in air with two electrodes placed 2% of the fish length apart
(approximately 1 cm) and sequentially moved from head to tail.
The spatio-temporal pattern of the discharge was analyzed by
means of the "multiple air-gap technique". The E M F generated by
each one of the four quarters of the fish body were simultaneously
recorded using the device described in Caputi et al. (1993). The fish
were placed in a four-compartment Plexiglass box (see Fig. 10) in
such a way that its body passed through successive conductive (2%
of the fish body length) and isolating media (23 %). Each conductive
compartment was connected to a high-input impedance unity-gain
non-inverting buffer amplifier, with their outputs connected to
differential amplifiers. The current generated by each studied portion produces a voltage drop across the input resistance of the
recording amplifier. This resistance was several order of magnitude
larger than the fish's R~. In such conditions the currents are negligible and the recorded voltages are equivalent to the EMFs. This
procedure avoids load sharing and permits one to obtain the values
of each portion independently of neighboring regions. Amplified
signals were either recorded on a pulse-code modulation video
cassette recorder or directly digitized (50 kHz sampling frequency)
and stored in a computer.
Bipolar field potentials were recorded using a differential amplifier while the fish was resting in the aquarium. In these experi-
636
A. Caputi et al.: The EO and EOD of R. rostratus
ments the electrodes were placed 1 cm apart and the rostral electrode was connected to the non-inverting input of the amplifier.
Partial curarization (d-tubocurarine, 1.5 Ixg" g-1) was used as
in Caputi et al. (1989, 1993) to complement anatomical-functional
data concerning the mechanisms subserving V4 and V5.
Results
Anatomical organization of the EO
The EO extends all along the body of the fish forming
an anatomical unit. However there are regional variations involving the number of electrocyte tubes and the
electrocyte innervation patterns. The gross anatomy of
the EO will be described first disregarding the distribution of dissimilar electrocyte types. The EO is composed,
at different percentages of the fish length, of different
number of electrocyte tubes (Fig. 1). In the sub-opercular
region (sample at 7% of the fish length) the EO consists
of one tube at each side of the midline. The number of
tubes increases dramatically toward the tail. Samples
obtained from the abdominal portion (15% of the fish
length) showed three bilateral tubes while samples from
the intermediate portion (obtained at 30% and 50% of
the fish length) showed respectively, four and five tubes
at each side of the midline. At the caudal portion (sample
obtained at 85% of the fish length) nine or ten bilateral
tubes occupy most of the cross section (occasionally an
asymmetric distribution of tubes was found).
Fig. 3. Camera lucida drawing showing at the left a RIE and at the
right a MCIE. In this case the tubes were disrupted but the electrocytes maintained their nerve attachments to AEN2. The thick
short arrow points rostrally, x 40
Fig. 4. Photomontage covering two focal planes of an osmiumstained MCIE. The two arrowheads indicate the axon bundles terminating in the caudal ridge ( C R ) and in the electrocyte margin.
The inset shows a semi-thin BMB stained section; the section level
is indicated in the photomontage (dotted line a - a'). The small arrow
indicates one thick axon contributing to the marginal innervation.
In both microphotographs (shown at the same magnification) the
thick short arrows point rostrally, x 50
.
.
.
.
jr-
A. Caputi et al.: The EO and EOD of R.
637
rostratus
Fig. 5. View of the marginal zone of an
osmicated MCIE at a higher magnification. Note the regular arrangement of the
preterminal axons that form a palisade
perpendicular to the electrocyte margin
(EM). The thick short arrow points rostrally, x 100
Fig. 6. Electronmicrograph showing a synaptic junction on the
marginal zone of a MCIE. Note that the nerve terminal (asterisk)
lies in a shallow depression of the electrocyte (E) plasma membrane.
x ll000
Fig. 7. Semi-thin section passing through the caudal ridge (CR) of
a MCIE. The small arrows indicate nerve terminals deeply encased
in the electrocyte plasma membrane. The medium size arrow indicates a preterminal axon; V blood vessel. The thick short arrow
points rostrally. • 380
In this species there are three types ofelectrocytes with
different patterns of innervation: RIEs, CIEs and doubly
innervated electrocytes. The latter receive innervation on
both their caudal and marginal faces (MCIE). The peculiar m o r p h o l o g y of these doubly innervated electrocytes
will be treated in m o r e detail below.
As described when dealing with the gross a n a t o m y of
the EO, the sub-opercular region contains a pair of M T
arranged one at each side of the midline. The MTs are
particularly long, extending f r o m the 7% to 100% of the
fish length; they contain CIEs. The three pairs of tubes
found in the abdominal region contain different kinds of
electrocytes: in addition to the MTs containing CIEs,
there are at this level a pair of SATs composed of RIEs
and a pair of intermediate tubes containing MCIEs (Fig. 2).
The intermediate EO region (from 30 % to 70 % of the fish
length) consists of several tubes (see above) containing
M C I E s and CIEs. The most ventral tubes are the MTs
found in the sub-opercular and abdominal regions and
contain CIEs; the other additional tubes (three to eight
at each side of the midline) contain MCIEs. The caudal
region of the EO (70 % to 100 % of the fish length) consists
of nine or ten bilateral tubes containing exclusively CIEs.
In s u m m a r y in the most rostral sub-opercular portion of
638
A. Caputi et al.: The EO and EOD of R. rostratus
mediate and caudal portions a pair of well-developed
PENs are found similar to those described in Gymnotus
(Trujillo-Cen6z et al. 1989).
the EO only a small number of CIEs occur; the abdominal
region contains three electrocyte types (CIEs, RIEs and
MCIEs); in the intermediate region only two electrocyte
types are found (a majority of MCIEs and a smaller
number of CIEs); finally the caudal region only contains
a large number of CIEs.
As shown in Figs. 2 and 3, RIEs and CIEs exhibit a
similar, conventional electrocyte morphology: they are
disk-like structures with only one innervated face.
MCIEs, however, are complex and deserve a more detailed description. Each MCIE has a concave rostral
surface and a convex caudal surface bearing a large
ridge-like projection (Fig. 4). Nerve terminals were found
concentrated on the caudal ridge and also forming a
palisade on the electrocyte margin. The marginal innervation derives from nerve fibers running in a rostrocaudal direction. These thick (20 gm) but lightly myelinated axons lie regularly arranged all around the electrocyte margin. Closer inspection shows that the nerve
fibers surpass the limits of the rostral surface and actually
terminate on the marginal surface, or even on the rind of
the caudal surface (Fig. 5). Synaptic junctions on the
marginal zone are not apparent on BMB sections but
their presence was confirmed by means of electron microscope procedures. They occur in shallow depressions
of the plasma membrane (Fig. 6). Conversely, synapses
on the caudal ridge are easily observed in BMB stained
sections; they are characterized by infoldings of the postsynaptic membranes in which the nerve terminals are
deeply encased (Fig. 7). In this respect caudal synapses
of Rhamphichthys electrocytes are similar to those observed in Gymnotus electrocytes. In the sub-opercular
region there is a pair of AENls that provide the nerve
branches innervating the caudal faces of the CIEs contained in the MTs. These nerves lie closely apposed to the
MT sheaths. At the level of the opercular aperture a pair
of AEN2s is added. They run between the intermediate
tubes and the SATs. In the abdominal region, branches
arising from the AEN2 innervate the caudal faces of
CIEs and the rostral faces of the RIEs. In the inter,
R/L)r
The spatio-temporalpattern of the EOD
Rhamphichthys rostratus emits brief pulses (2.5 ms duration) at a very constant firing rate (50+ 0.2 Hz, 23 ~
Fig. 8A). The electrical behavior was quite similar to that
50 mV
L
20 m s
V
b
3
2
1
0
-1
-2
-3
V3
1 ms
V4
0 dB
-50
-100
-150
-200
-250
I
0
I
i
KHz
E M F - Ri. I
Fig. 9. Sketch of the circuit used to
measure V and I generated by the fish in
the air-gap condition (upper left.). Plots
correspond to the main EOD deflections
(V1-2, V3, V4) of the EOD. Each plot
shows the relationship between I and V
obtained with different load resistances
(RL). The E M F is the ordinate intercept;
the Ri is the slope of the line. The resulting values are shown at the upper right
V3
V3
V1-2
2
2
0.2
1
. . . . .
0.02
I
Fig. 8. a Four EODs of R. rostratus recorded head-to-tail in water.
b EMF of a single EOD with its different wave components, e Power
spectral density of a single EOD (dB referred to the total power).
fish
0.00
I
5
EMF (V) Ri (Rf~)
0.32
10.4
2.65
10.7
2.72
10.5
V1-2
V3
V4
0.0
I
,
V-
V 0.4
I
3
0.04
mA
o
o.o
9
,
0.1
9
,
0.2
9
,
0.3
mA
0
0.0
0.1
0.2
0.3
mA
A. Caputi et al.: The EO and EOD of R. rostratus
639
described by K r a m e r et al. (1981) in an undetermined
species of the same genus.
The EOD shows four main deflections (head to tail
records) here referred to as the V 1-2 complex (see following paragraphs), V3, V4 and V5 (Fig. 8B). The power
spectral density of a single EOD has a peak at approximately 1 kHz and spreads between 10 H z and 5 kHz
(Fig. 8C).
The EOD wave components have dissimilar E M F
amplitudes and dissimilar spatial origins. As shown in
Fig. 9, the V1-2 complex is ten times smaller than V3 and
V4. The amplitude of V5 is similar to that exhibited by
the V1-2 complex. The Ri is about 10.5 kf~ for all wave
components. Thus, the maximum power delivered to the
load (0.6 mW) occurs when the resistance is about 10 kf~
[theorem of maximal transference of energy, Donaldson
(1958)]. This value is similar to the resistance measured
between two electrodes immersed in fresh water 50 cm
apart. Figure 10 illustrates the spatial distribution o f the
different EOD wave components: the spatial domain of
the V1-2 complex extends from 10 to 75% of the fish body;
V3 is generated almost all along the fish (5-100%), while
V4 and V5 arise from the caudal 50 %. The amplitude of
all wave components increases monotonically in a rostrocaudal direction along its spatial domain. As shown in
Fig. 10, the peaks of the EOD wave components, recorded from different body portions are not synchronous.
F o r any given wave component the peak at caudal level
is delayed with respect to the peak at more rostral levels.
This phenomenon can be described as a head-to-tail
wave that travels at a mean speed of 2 k m . s-1.
Anatomical-functional correlation
values of each portion independently of neighboring regions. The
lower trace corresponds to the head-to-tail EOD
In topological correspondence with the anatomical organization four functional portions were identified. Figure 11 shows a summarized view of the anatomicalfunctional organization of the EO o f R. rostratus combining electrocyte-type distribution (represented by different shades) and four air-gap (2% fish length-width)
recordings. Single-air-gap recordings were sequentially
obtained all along the fish length and four representative
examples corresponding to the four anatomical regions
are shown. The EOD recorded from the opercular region
only shows V3. As described in the anatomical section,
at this level the EO contains exclusively CIEs.
The air gap studies (Fig. 1 l) and the rostro-caudal
series of bipolar field potentials obtained close to the fish
(Fig. 12) revealed that the V1-2 complex consists of two
wave components with overlapped spatial and time domains. There are variations in the EOD waveform when
air gap and bipolar field potentials are compared. This
results from the different load applied to the EO. In the
air gap there is no load, whereas in water a complex
distributed impedance loads the generator. Wave component amplitudes and peak times depend not only on
the fish electrical parameters (local EMF, tissue resistance and skin resistance) but also on the loading media
(water resistance, border conditions, etc.). Field poten-
Fig. 11. This semi-schematic drawing correlates different EO portions with samples of the regional EODs (air-gap recordings from
1-cm portions at 10, 20, 60 and 85%). It also illustrates the distribution of the different electrocyte types along the EO (RIEs diagonal
shading; MCIEs stippled; CIEs vertical shading). Recordings at
10%, where the EO contains only CIEs, show a monophasic headpositive deflection followed by a small ripple. At the abdominal
level, where the three types of electrocytes occur, the V 1-2 complex
appears (arrow). In the intermediate region (lacking RIEs) the
regional EOD lacks V 1 while V2 becomes relatively larger. At 85 %,
where the EO is composed of a large population of small CIEs, the
regional EOD shows a single neurally elicited wave (V3) followed
by the non-neurally elicited components (V4-V5). Information concerning individual tubes is only provided for the rostral portion
(note that the EO has been represented as discontinuous and that
the natural proportions have been not maintained). In the intermediate region all tubes containing MCIEs are represented as a
whole; in the caudal 25% all tubes contain CIEs and are also
represented as a whole. Vertical calibration bar corresponds to 25,
50, 150 and 250 mV, respectively. Horizontal calibration bar: 3 ms
1012v
~
~>
0.24V
I0.70
v
1.40 V
Fig. 10. Spatiotemporal pattern of EMF along the fish body. The
four upper traces correspond to successive fish body quarters as
recorded in the four gap chamber schematized at the left. This
procedure avoids load sharing and permits one to obtain the EMF
640
%
40
30
25
20
15
A. Caputi et al.: The EO and EOD of R. rostratus
i
E
mV
50 i ,,.o- b.."
?"...............
tl
-10
%
0
[]
tl t2
a
q,00mv
i'
0 ~-%.........................;~. . . . . . .
1 ms
20mY ~ ',, ~
"151 ~,~'"
%
t2
16o%
Fig. 12. Field potentials recorded in water facilitated identification
of the two wave components of the V1-2 complex. V1 and V2 have
overlapping temporal and spatial domains. The traces at the left are
successive bipolar recordings (between 15 and 40% of the fish
length) of the EOD in water. Plots at the right show voltage values
obtained at T1 (circles) and T2 (squares) versus percentage of fish
length. V1 originates between 15 and 25% (upper plot) while V2
originates between 10 and 75% (lower plot)
tials taken close to the fish may be interpreted as the
effect through the media of the summated currents that
flow between the electrode locations. In the absence of
other sources these currents are generated by the EO
discharge. Therefore, such records were used only as a
qualitative index of the activity of the EO. The place
where the amplitude of each wave component (V1 or V2)
diminishes indicates the place where the transcutaneal
currents change direction. The reversal points indicate
the place where transcutaneal current flows attain their
maxima and are good indicators of the limits of the
generators. The reversal points were close to the caudal
limit of the spatial domains of RIEs and MCIEs and also
close to the caudal limit of the spatial domains of V1 and
V2 (recorded in the air-gap).
The earliest wave component (V1) occurs exclusively
at the abdominal region (the region in which RIEs occur), whereas the spatial domain of the other wave component (V2) extends from 10% to 75% of the fish body
(the region containing MCIEs). As in the sub-opercular
region, V3 results from the activation of the caudal faces
of most electrocytes.
Anatomical data have revealed that the tail region of
the EO is composed, as in G. carapo, of a quite homogeneous population of CIEs. In the latter species V4 is the
consequence of the activation of rostral non-innervated
faces. A similar situation occurs in R. rostratus. The
activation of rostral non-innervated faces should arise
from action currents spreading from the caudally innervated faces. This hypothesis was tested by recording the
EOD of the caudal 10% in partially curarized fish. The
temporal evolution of the D-tubocurarine effect was
characterized by describing the two stages represented in
Fig. 13. The initial stage showed a disorganization of the
EOD accompanied by a reduction in its amplitude. The
disorganized EOD consisted of an early positive deflection followed by multiple positive-negative discharges
that varied in number and amplitude in the course of
time. In the second stage, the EOD remnant became
stabilized. Curare dramatically diminished V3 and
13
4 mV
2 ms
Fig. 13. a-c. Curare dramatically diminished V3 and abolished V4
and V5: a control EOD; b and e curarized animal. After a period
characterized by variable patterns of desorganization (example in
b), the EOD became stabilized, small and monophasic (e). The
biphasic peripheral nerve activity (arrow) remained unchanged. To
compare this activity in the same fish before and during curarization, an equally magnifiedcontrol recording (inset in e), is included
abolished V4 and V5. The EOD remained as a positive
deflection resembling in shape and time-course and endplate potential. Figure 13 also shows a small biphasic
(1 mV) deflection (arrow) that did not change in amplitude or shape and may correspond - as demonstrated
in G. carapo (Caputi et al. 1993) - to the activity of the
PEN and its branches. Control and curarized animals
showed the same delay between nerve and electrocyte
activity.
Discussion
Investigations on the functional organization of the electrogenic system can be based on two complementary
strategies: a) the detailed study of a single species concentrating efforts to cover different levels of analysis from
behavioral to cellular events, and b) the initiation of
comparative studies including less known species and
families. The second approach allows one to identify
general evolutionary traits and variations from the
general structural and functional pattern (Bullock 1984,
1986, 1993).
The idea that the electrocyte innervation pattern is
important for determining the EOD waveform was introduced by Bennett and Grundfest (1959) in their
pioneer studies on G. carapo. More recently we advanced
the hypothesis that waveform generation may be dependent upon the electrocyte innervation characteristics, the
properties of the neuroelectrocyte junctions and the size
and spatial distribution of electrocytes along the EO
(Caputi et al. 1989; Macadar 1993). This hypothesis was
confirmed by means of single electrocyte recordings (Macadar et al. 1989). The present study dealing with the
poorly known Rhamphichthys has revealed the occurrence
of three types of electrocytes (CIE, RIE and MCIE) with
different innervation patterns. Within the conceptual
641
A. Caputi et al.: The EO and EOD of R. rostratus
framework developed in our previous studies the spatial
distribution of these three electrocyte types have been
related with the different wave components of the EOD.
The EOD of R. rostratus consists of four main deflections. A detailed analysis of the first head negative deflection (V1-2 complex) has revealed that it actually consists
of two wave components. However, the striking topological correspondence between the spatial domains of these
wave components and the distribution of the different
electrocyte types suggested that V1 may originate from
the activities of the abdominal RIEs while V2 may originate from the more widely distributed MCIEs. V3 probably originates by the activation of the caudal faces of
most electrocytes. It is important to emphasize that at the
opercular region where only CIEs occur the regional
EOD only shows V3 followed by small ripples not elicited by neural command (they were abolished by curare).
Since V4 and V5 were abolished by curare it has been
inferred that they are not directly elicited by neural command. Moreover, curarization experiments revealed that
peripheral nerve activity occurs before V3. Neither in
normal condition nor in curarized animals were signs of
nerve activity preceding V4 or V5. Previous investigations on G. carapo (Caputi et al. 1989; Macadar 1993)
revealed that the last EOD wave component (V4) is not
neurally elicited: it originates by the invasion of rostrally
innervated faces by action potentials triggered on the
caudal faces. Likely, V4 and V5 of Rhamphichthys have
been interpreted as the successive invasion of action
potentials previously originated at opposite electrocyte
faces. This kind of reverberating phenomena have been
occasionally observed in G. carapo under experimental
conditions (Caputi et al. 1993). In the tail region electrocyte size diminishes while electrocyte density increases. Both features may favor electrotonic activation
of electrocyte membranes by outward currents generated
in the opposite faces of the same and neighbor electrocytes.
With respect to the progressive delay of the occurrence of the V3 peak along the EO, the same feature was
reported in G. carapo (Caputi et al. 1993), and in Electrophorus by Coates et al. (1940) and Albe-Fessard and
Martins-Ferreira (1953). It can be interpreted as a fast
excitation wave travelling along the EO. Even though
accurate calculation of the wave speed is difficult, it
should range between 700 and 2000 m 9s-1 (when it is
assumed that the peak of most rostral V3 coincides with
the V2 peak a lower speed is obtained; when it is assumed
that V3 peak is not affected by its summation with V2 a
high speed is obtained). In no case are these values
compatible with nerve conduction velocities (Macadar
1993). Other neural mechanisms, as those proposed by
Bennett (1971) and described by Lorenzo et al. (1990) in
G. carapo, should also occur in Rhamphichthys. Figure
11 shows the summarized view of the EO organization
of R. rostratus, including the corresponding regional
EOD waveforms.
The occurrence of doubly innervated electrocytes has
been reported only in G. carapo (Szabo 1960, 1961;
Trujillo-Cen6z et al. 1984), but the present findings indicate that they also occur in another taxon. In the case
of Rhamphichthys these electrocytes show the following
distinctive features: a) the nerve fibers arriving from the
rostral side form a regular palisade all over the electrocyte margin; b) synaptic junctions occur on the marginal membrane; c) the caudal face of each MCIE bears a
large ridge receiving most of the caudal innervation.
Anatomical-functional correlations suggested that
MCIEs may be the electrogenic units that give origin to
V2 and also contributes to the genesis of V3. As it occurs
in G. carapo the neural activation of caudal faces may
generate V3. Concerning the mechanism subserving V2
there are evidences indicating that it does not result from
action potential invasion from caudal faces (V2 occurs
before V3). It should be noted that the amplitude of V2
is ten times smaller than V4 in the region of the organ
where mainly MCIEs occur (50-75 % of the fish length).
This feature indicates that not all the electrogenic capability of rostral faces of MCIEs is involved in V2 generation. However, the existence of a highly organized neural
palisade on MCIE margin may indicate that such regular
arrangement of nerve terminals is involved in the generation of V2. It is an open question whether V2 results from
summation of peripheral neural activities (as occur in
Apteronotidae), from the graded activation of rostral
faces (as occur in lateral abdominal electrocytes of
G. carapo), or by the combination of both mechanisms.
Electrophysiological recordings at the cellular level are
required to explore the membrane mechanisms supporting V2 generation.
Acknowledoements. We thank Dr. D. Lorenzo for his comments and
useful suggestions. This investigation has been partially supported
by: The European Economic Community (Contracts No.
CI1"-CT92-0085; CI1"CT901-9861) and the Comisi6n Sectorial
de Investigaci6n Cientifica de la Universidad de la Repfiblica.
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