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ELECTRIC FISII INVESTIGATION

Final Report

Contract

8 March 1973

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ILLUSTRATIONS

Page
ure
1 Mauthner cells 4
2 Electrorcccptors 6
3 Cranial nerves of Electophorus 8
4 Microelcctrode amplifier 13
5 Microeloctrode amplifier: schematic diagram 13
6 Loss in -dB with increasing frequency 14
7 Loss in -dB with increasing repetition rate 15
-8 Device for sharpening metal electrodes 17
9 Device for applying silver chloride 17
10 Potentiometric device 19
11 Device for measuring microelcctrode resistance 19
11A Amplification factor, square wave 20
11B Amplification factor, sinusoidal wave 21
11C Recordings at different square wave 22
11D Recordings at different square wäve 23
27
12 Adiustable Lucite Tray
13 Instrumentation for Anaesthesia 27
14 Solution 1:10000 in water on Sternarchus albifrons 29
15 Sternarchus albifrons #7 normal elcctrical activity 31
16 Sternarchus albifrons #7 in ancsthetic tricaine methane 31
sulfonate, 1 minute
17 Sternarchus albifrons #7 in anesthetic tricaine mcthane 31
sulfonate,2 minutes
31
18 Sternarchus albifrons #7 in anesthetic tricaine methane
sulfonate, 4 minutes
19. Sternarchus albifrons #7 in anesthetic tricaine methane 32
sulfonate, 5 minutes
20. Sternarchus albifrons recuperating, 5 minutes 32

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32
1 Sternarchus albifrons recuperating, 8 minutes
32
:2 Sct-up for microelcctrode
13 Sternarchus albifrons No. 1 just before anaostliesia 33
24 Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 33
(2 min.)
25 Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 34
(13.5 min.)
Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 34
26
(37.5 min.)
27 Sternarchus albifrons No. 2 in aquarium water 36
28 Sternarchus albifrons No. 2 in MS-222, 10 minutes 36
38
29 Set-up for recording
38
30 Close-up of the set-up for recording;
31 Sternarchus alhifrons after d-tubocurarine injection 40
32 Ampullary, tonic electroreceptors 40
41
33 Set-up for recording
34 Anaesthetized, curarized Sternarchus albifrons 41
43
35 Microelcctrode amplifier
44
36 Preamplifier
37 Synchronous tonic electroreceptor recording 44
3S Nonsynchronous phasic electroreceptor recording 44
44
39 Gymnarchus niloticus Cuv. #2 electric activity
40 Gymnarchus niloticus Cuv. #3 electric activity 46
41 Gymmarchus niloticus Cuv. #2 electric activity 46
46
12 Gymnarchus niloticus Cuv. #2 electric activity
46
13 Gymuarchus niloticus Cuv. #3 electric activity
47
44. Gymnarchus niloticus Cuv. #3 electric activity
15 Liyn narehus niloticus Cuv, f3 clcctric activity 47
46. Gymnarehus niloticus, Cuv, (baby) - transversal cut 48

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Figure

Page

47 Gymnarchus niloticus Cuv. - air bladder and spinal cord 49 i
48. Gymnarchus niloticus Cuv. - spinal cord 49
49
49 Gymnarchus niloticus Cuv. - brain
50 Delafield - Harris hematoxylin staining method 50
51 Malaptcrurus electricus 52
52 Malapterus elcctricus on the scale 53
53 Device for measuring the voltage 53
54 Discharges of the electric organ of Malapterurus electricus 54
56
55 Tonic, ampullary electroreceptor
56 Phasic, tiberous electroreceptor 57
57 Tonic electroreceptor 59
58 Phasic electroreceptor 60
61
59 Ampullary tonic electroreceptor
60 Stimulus recording 61
61 Tuberous phasic electroreccptor 61
61
62 Stimulus recording
63 Electroreceptors of Gymnarchus niloticus 62
64 Undervater pattern recognition system G3
65 Recording from an anaesthetized, curarized 66
Sternarchus albifrons specimen
66
66 Microelectrode recording
67 Gyınuarchus niloticus - photo 70
68 Gymmnarchus niloticus - placcment of elcctric 71
transmitting and recciving organs
72
69 Fish Laboratory A
72
70 Fish Laboratory B

y

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SUMMARY

The electric organs of Sternarchus alhifrons, a South Amcrican fresh water weak electric fish, have becn studicd with emphasis on electroreceptors. The morphological and physiological characteristics of electroreccptors, ampullary and tuberous, a. e discusscd. Spccial instruméntation required for the role of these elcctroreceptors in pattern recognition has been developed. we have recorded with microclectrodes the autonomous autorhythmic electrical activity of the tonic asynchronous ampullary electroreccptors of the South American weak fresh water electric fish Sternarehus albifrons. We have also recorded the electrical activity from the phasic tubérous electroreceptors and of the synchronous ampullary electroreceptors of the same electric fish, Sternarchus albifrons. Preliminary measurements have been made. The electric discharge of Malapterurus electricus, an African fresh water strong electric fish, has becn measured in and out of water. The autorhythmic activity of the ampullary electroreceptors has been demonstrated. We obtained somc specimens of the African weak fresh water electric fish Gymnarchus niloticus. They are supposed to be the most sensitive of all the weak electric fishes known. Together with two specimens about one foot long, we received a number of baby Gymnarchus niloticus about two inches-long. The baby electric fish were infected with a Saprolegia fungus and could not be saved, but we fixed a number of them in buffered formaldehyde and one of them has been cut and mounted in paraflin for histological studics of the electric organs. Preliminary measurements have been made on the communication capability of adult Gymnarchus niloticus. A study of thc ancsthctizing effect of tricaine-mcthancsulfonate (MS222 = FINQUEL) on Sternarchs albifrons has been undertaken by plotting timc for the anesthesia and recovery for different specimens.

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A study of an anaesthotic which docs not affect thc elcctric fish's electric organ pulse repctition rate is presented.

Also, the effect of D-tubocurarinc and the counter-effect of ncostignine has been assessed for Sternarchus albifrons. Finally, some improvements in the microelectrode recording instrumentation have been made.

The electric organs of Stornarchus albifrons, a South American weak fresh water electric fish, have been studied with emphasis on electroreceptors. Recordings have been made from the asynchronous tonic as well as the symchronous tonic and the asynchronous phasic electroreceptors. The electroreceptors are part of the complex lateralis line system of the electric fishes.

The other lateralis line system sensory receptors, like mechanical receptors and displacement receptors, have been discussed as part of a general hybrid pattern recognition system of the fish. A passive hybrid underwater pattern recognition simulation system has been advanced.

A simulating model concept could be established for underwater pattern recognition through electric sensory receivers and electric fields. More histological work is needed to establish the rlationship between different elcctroreceptors and their innervation, This is also needed for a realistic simulation system of the underwater pattern recognition ability of the elcctric fishes.

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I. INTRODUCTION

In one of our provious reports, we described the morphology of the electric organ 1 of Sternarchus albifrons, a weak fresh water electric fish from South America. We mentioned that the electric transmitting organ of Sternarchus is derived from nervous tissue and not from modified muscle tissue like the majority of other electric fishes. This is making Sternarchus different from other electric fishes: it has a very high signal rate and the signal is phase and amplitude modulated.

The form, rate, and amplitude of the signals emitted by the electric fishes are as diverse as the forms and sizes of these fishes. Some of the weak signals are used to locate objects or animals in their environment, or for navigation, species recog-2
nition, and communication. The strong electric discharges serve for offense or 3
defense. Watanabe and Takeda“ investigated the effect of a-c current with a frequency close to the electrical signal emitted by Eigenmannia. When the applied pulses came within ± 3 to 4 pps of the one emitted by Eigenmannia, the fish would change its rate by 4 to 5 pps in a direction which increased the pps separation. Increasing the frequency of the applied a-c current in 1 pps increments caused the fish to shift its frequency correspondingly until it reached about 6 to 7 pps over its normal rate, when it would revert to its original rate. We obtained similar results in experiments with Sternarchus albifrons, but the applied a-c signal was within 0.5 cycle of the signal of thc fish, demonstrating how specific the applied signal must be to elicit a change in the Sternarchus signal rate: ± 0.5 cycle is the range of selectivity of tho fishes'electrical transmitting-roceiving system. The fact that many fresh water and sea water eleciric fishes have never been studied may prescnt some difficulties in obtaining spccial kinds of electric fishes, and thcir care may not be an easy task. There are, however, enough species to enable many experiments.

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Very little is known about the clcctrical activity of marine clectric fishes except Torpedo and some rays. Narcine Barziliensis is the only known marine electric 4
fish having two different electric organs. Bemnett studied the mode of operation of the electric organs of Raja eglantaria, a marine elcctric fish, and of the fresh watcr fishes Hypopomus and Sternopygus and compared them with Narcine, Mormyrus, Steatogenys, Gymnorhamphychtis, Malapterurus, Gymnotus carapo, and Electrophorus elcctricus.

The main objects of this study were the form, innervation, and physiology of the electroplates forming the electric transmitting organs. The electroplates of Hypopomus, Gymnotus carapo, Malapterurus, and some Mormyridae have the same surface area and produce spikes during discharge. The electroplates of Sternopygus and possibly Eigenmannia have two peculiar characteristics: there is a steady potential on which pulses are superimposed, and the resistance of the electroplates is similar at the peak or between the spikes. The electroplates are of the type with a slow depolarization.

In Hypopomus, Malapterurus, and most of the Mormyridae, the innervation of the electric organs is through stalks. The stalks may serve to amplify depolarization until it would be able to invade the body of the electroplates.

The discharge rate and duty cycle have been compared for electric fishes like Sternopygus (rate = 50/sec) and Eigenmannia (rate = 280/sec) with the Sternarchidac (max. 1500/sec). Compared with mammalian central nervous systems, peak frequencies of the electric organs are not greatly different. The Renshaw cell can discharge impulses at a rate of $1 4 0 0 / \mathsf { s e c } ^ { 5 } ;$ , and neurons in the sensory path some-6
times produce bursts at a rate of about 1000/sec.

In an examination of discharge pattern and organ function studies, it was noted that Grundfest defined two groups of clectric fishes: those that emit signals at a constant

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rate, and those that emit pulses with a variable rate. For example, Gymnarchus, Sternopygus, and Eigenmannia are in the constant rate group; Electrophorus, Gnathonemnus petersii, Steatogenys, and Hypopomus belong to the variable rate systems, their environment, and their behavior. No one investigated their evolution, very little is known about their mating or birthplace, and no one has reported the breeding of electric fishes confined in water tanks.

Sternarchus albifrons also has two kinds of electroreceptors: tonic and phasic, and they are autorhythmic. These electroreceptors are sensitive to movement and direction. The phasic electroreceptors seem to be related to informatian regarding movement of objects near or around the fish.

Accepting the principle of pacemaker activity in the brain, it seems that there are only a reduced number of command nuclei acting on the electric transmitting organ. It is also reasonable to assume that electrically mediated positive feedback must be present; chemically mediated transmission would be too slow for the repetition rate of transmission which can attain under certain circumstances over 1, 300.

Mauthner cells of lower vertebrates (Figure 1) can be considered single cells 8,9
command system for the, axial musculature on either side of the body . In the
hacketfish each Mauthner fiber activates the muscles depressings both pcctoral fins
and these cells thus constitute a bilateral command system for the depressor 10,11
muscles. For explaining the pacemakers action of the command nuclei in the brain
of the mormyrid electric fishes a mutual excitation with positive feedback has been
proposed. This theory would not work for the Mauthncr cells. There is a crossed
inhibition between the Mauthner cells in the brain of the goldfish and it could equally
be effective in an electric organ system. There is a requirement of high speed
nf transmission in synchronized systems like many of the electric transmitting
organs. This has been useful in predicting sites where transmission has been

3

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Figure 1. (a) Schematic drawings of the location of the Mauthner cells (from Furukawa and Furshpan")

(b) The principal circuitry involved in Mauthner reflex (only the V111-th nerve conncctions and collaterals deriving from the excited Mauthner ccll are shown).

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electrically mediated. Positive fcedback may be a "sine qua non" requirement for the pacemaker nuclei of electric transmitting organs. There is proof of positive feed-12,13 back in the mutual inhibition system of arthropod compound eyes

Th; neural systems controlling electric organs have provided a largc number of exi-mples of electrically mediated transnission, which meets the functional requirement for rapid communication between cells. This mode of transmission also proves to be able to mediate many functions often considered as restricted to chemically mediated transmission. The corrclatfon between morphologically close apposition and electrotonic coupling was considerably strengthened by thc work on electromotor systems. This correlation helps to validate morphological identification of electrical transmission in other systems where electrophysiological analysis is not so simple.

It is not known whether there is any relevance to higher systems of the organizational principles deduced from electric organ systems. The next level of analysis of the electric organ systems may be no easier than the study of less specialized systems that are of more gencral interest. Some knowledge is being obtained of afferent pathways from electroreceptors in weakly electric fish which have important inputs to the electric organ control system. Both operant and respondent conditioning of the control system can be obtained and conditioned response latency can be very short. It is not unreasonable that the complete neural pathway of the conditioned response could be obtained in these cases. The central connections are still minimally explored; one knows what goes in and one can go from the electric organ several synapses antidromically. The rewards for filling in the gap would be great, and prospects for at least some progress are bright.

la Sternarchus albifrons electrorcceptors are distributed over the entire body. The phasic tuberous receptors are very numerous as compared with the tonic ampullary receptors (Figure 2). The density of reccptors is greaiest in the head region and

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Tonic, Ampullary Receptor

Phosic, Tuberous Receptor

Figure 2. Anatomical diagrams and equivalent eircuits of clectroreceptors in fresh water clectric fish: (a) tonic, ampullary receptor and (b) phasie, Auberous reeeptor. Diagrams are shown with the external medium to the top. The skin and wall of receptor cavities are shown in cross so ction as lines. The opening to the exterior of phasic receptor cavity is shown as oceluded by a porous mass. The nerve fibers innervating receptor cells are indicated.

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falls gradually toward the posterior end. There are minor morphological subdivisions within the phasic and the tonic receptors, but no physiological corrclations have been yet obtained14, 15,16

In Figure 2 the equivalent circuits of tonic, ampullary and phasic, tuberous organs is shown. Cross sections through the receptors are shown with the extemal medium to the top. The skin and walls of the receptor cavities are represented by lines, innervation of the receptor cells is indicated.

The electroreceptors over the entire body are innervated by the anterior lateral line nerves, a large branch of which runs posteriorly to join the posterior lateral line nerve just behind the head17 (Figure 3). The posterior lateral line contains only 18,19 mechanoreceptive fibers which come from free neuromasts and canal organs

The receptor cells of tonic receptors appear to behave very nearly like linear elements; that is, their membranes have fixed internal potentials, resistances, and capacitances. They are, in a sense, electrically inexcitable, and they differ markedly in this respect from phasic receptor cells. There is evidence for chemically mediated 20 transmissions at tonic receptors of gymnotids ". The morphological characteristics of the snyapse are those typical of chemically mediated transmission. A strong brief anodal stimulus produces an evoked response long outlasting the stimulus. Then there is a synaptic delay between the initiation of the impulse and the nerve impulse (between 0.5 and 1.5 msec). Mormyrid tonic receptors are similar to those of gymnotids and Gymnarchus niloticus tonic receptors are morphologically similar,

The relationship between thc different elcctroreceptors of Sternarchus aIbifrons in pattern rccognition has not as yet been studied. The present study developed special instrumentation required for investigating the roles of these elcctroreceptors in pattern recognition and obtained preliminary measurements of electrical discharges from Malapterurus clectricus in and out of water.

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Figure 3. liorizontal projection of the cranial nerves of Electrophorus. Note the small size of the brain. Some nerves are indicated by fhe corresponding numbers. L.A. nervus lateralis anterior; . LP nervus lateraíis posterior; Oc-Sp, occipito-spinalis nerve.

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In our experiments, the perception of objects by weak clectric fishes by way of the discharge of their electric organs (transmitting and receiving) has bcen demonstrated. Other authors22 have shown that weak electric fishescan be trained to distinguish betwveen conducting and nonconducting objects placed in the water.

For this kind of reception, two possible modes of action at the level of spccific rebe conveyed by the frequency of the sensory impulses dependent on the pulse of the electric organ discharge whereas, according to the second hypothesis, thc time relation (the phase) between the electric discharge and the following sonsory impulse would play an important role for the sensory coding. From our experimcnts with Sternarchus albifrons, a South American weak fresh water electric fish, we concluded that apparently neither of the above proposed mechanisms are operating in this fish. The intensity of current flowing at the level of the receptor is coded by the number of impulses elicited by each electric organ discharge. St:rnarchus albifrons can discharge at rates higher than 1000 per second and the sonsory impulses of the receptors can follow their discharge rate.

In some mormyrids 24 and gymnotids, however, modulation of the rolation electric- 25 organ pulse-sensory impulse may be used for elcctrosensory coding. In mormyrids, also, the change in intensity of the electric field may be codod by charging the latency between the electric organ pulse and the sensory receptors' impulse. In mormyrids, it may attain values up to 9 msee, whereas in gymmotids it has been found not to excced 1-2 msec.

It has been previously mentioned that in Sternarchus albifrons the clc::roreceptors are distributed over the entire body of the fish and that the ampullar: conie receptors are more numcrous than the tuberous phasic receptors, We recorder: ¿en the autorhythmie electrical activity of the nonsynchronous tonic, ampullary c: :etroreceptors of Sternarehus albifrons. The impulses were irregilar around a repe-ion rate of

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betwecn 100 and 300, with an amplitude of around 2.5 mV. The impulsc duration was around 200 microscconds. Therc are other types of tonic receptors which are synclronous.The phasic units are nonsynchronous.

The nonsynchronous tonic receptors scem to react independently from the transmitting electric organ. They react to any objects brought near the fish at a certain range. Thc rccording shown in our final report is made from such type of receptors.

As previously mentioned, some fresh water weak electric fishes have the ability of perceiving objects, thoir movement and direction, and also to determine some characteristics of these objects (such as conductivity). For this underwater pattern recognition, they utilize both their electric transmitting organs and their electroreceivers. Obviously some other sensory perception receivers of the lateralis line may be involved such as: free neuromasts and the cupula type of lateral line receivers; the auditive system may play a role also in this pattern recognition. Some of the electric fishes are blind or have vestigial eyes; othcrs have good vision and it is certainly used in the recognition process.

It scems that a hybrid system made of a diversity of sensory receptors is used by the electric fishes to locate and identify objccts and fishes of the same or different spccies and also prey or predators. They use littlc electrical energy for this and tho distance involved is considerable - it may attain, under certain circumstances and for one specific sensitive species (Gymnarchidac), over a mile.

The strong clectric fishes (Elcctrophorus, Malapterurus, Torpedo, and Astroscopus) can incapacitate and kill their prey with their discharge of the powerful alectric orgn used only for such purposes. I they have a detecting and locating systen, they use a different low power clcctric transmitting than thc main powerful electric organ (i.e., Electrophorus). The meaning of this is that there is no high power requirement for the pattern recognition system of clectric fishes. The electric

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sensory receptors are very sensitive, have a high discriminating capacity, and use an clectronic procossing system based on a nultiple degree of frcedom modulating and coding system; they are also jamming resistant.

The importance of such an underwater pattern recognition system cannot be overemphasized. The simulation and modeling of it can be achieved once the parameters of the different sensory receivers have been estalished, physical analogs derived, and models devised.

By and large,the communication and códing system of electric fishes has been 27
demonstrated through simulation'. The electroreceptors of Sternarchus albifrons,
a fresh water weak electric fish, have been studied, and for two different kinds,
physical analogs have been proposed.

Because of our previous findings that the anaesthetic "MS 222(Finquel)"used by most researchers of electric fishes affects the frequency of the impulses emitted by the clectric organs in a nonuniform way, we investigated a number of different anaesthetics and found one which does not affect the frequency of the impulses or their amplitude.

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I. INSTRUMENTATION

The recordings of the autorhytmic electric signals from electroreceptors nced special instrumentation. The signals are of low voltages: less than 1 mv, and they need a large bandwidth. The diameters of some of the electroreceptors are of the order of microns. There is the need for an insulated microelectrode with a tip of one micron, with a reasonable low resistance, possibly less than one megohm, responding from DC to several kHz with low distortion and enough rugged to withstand some bumping by the fish. It has also to be of a nonpolarizable type. We developed a microelectrode with all these characteristics and it is described later in this chapter.

In our proposal for the continuation of the investigation of electric fish we mentioned that we developed a sensitive low noise solid-state microelectrode d-c amplifier (Figures 4, 5). We modified them having now a Motorola MC 1531 input stage and a Motorola MC 1431 output stage. Graphs show the repetition rate versus amplification factor for square wave and frequency versus amplification factor for sinusoidal waveforms with a 100 kiloohms, 1 megohms, and 5 megohms input resistance (Figures 6, 7).

We give a description of the method used to produce a suitable microelectrode for recording electric signals from the electroreceptors of Sternar chus albifrons.

29 10 31 described glass insulated platinum microclectrodes; Grcen and Grundfest, et al used stainless stecl clectrodes. The steel and tumgsten electrodes have a fairly high resistance (20 to 100 mcgohms) and all, including the platinum electrode, are polarizable. Donaldson describes a multitude of mieroclcctrodes such as silver-silver chloride, 32 platinum-platinum chloride, and others. Silver-silver chlorides are very convenient

12

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13

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elcetrodes but have a high resistance. In order to lower the resistance, it is advisable to cover the silver-silver chloride clectrode after the electróde is insulated with insulex lacquer, with płatinum black through an electrolytic process and after this to add a new silver-chloride coating. The tip of the electrode can be anywhere between 0.5 and 1 micron in diameter.

Take a 50 ml beaker and fill it with conc. H Cl and cover it with Xylene (about $1 / 2 " >$ , Use a carbon rod for one electrode (spectroscopy carbon rods are suitable) and connect it to a variable source of A.C. current from a variac and a bell-type transformer (app. 7 to 12 v). Connect the low voltage leads to an A. C. voltmeter. Use about 5 to 6 volts to sharpen the silver wire #36 gauge (any necessary length) by moving it up and down for about one minute (Figure 8). Decrease the voltage to between 2-3 volts and move the electrode rapidly for 30 seconds up and down. Have 50 ml beakcrs filled with: (1) saturated sodium carbonate solution $\mathfrak { N a } _ { 2 } \mathfrak { c o } _ { 3 } )$ (2) acetic acid 1% in $\mathfrak { H } _ { 2 } ^ { \mathrm { ~ O ~ } ( \mathsf { C H } _ { 3 } \mathbb { C O } ) } \mathfrak { z } ^ { \mathrm { ~ O ~ } , \mathrm { ~ O ~ } , }$ (3) Ethyl-alcohol 200 proof, (4) Xylene. Move electrode after sharpening process fron No. 1 through No. 2, 3, and 4, agitating à few times the electrode in the liquid. Check under microscope with a microfilar for sharpness; if not sharp enough, operation II (2-3 volts) and the cleaning from 1 to 4 should be repeated.

If sharp enough, take a 50 ml beaker and fill it with Na Cl 1% solution in distilled $^ { 1 1 } { } _ { 2 } ^ { 0 . }$ Use a silver wire (No. 18 to 22 gauge) as an electrode (cathode) and connect it vith a D.C. source (power supply) of between 1 to 2 volts. The positive end should be connccted to the microclectrode. Hold it for 30 seconds in the H Cl solution. Reverse twice the polarity for the same amount of time (Figure 9). Wash the electrode in distilled water for two minutes. Insulate the electrode with insulex laequer. For drying, set the microclcctrodes with the tip up. Dry for 24 hours. The tip should be clean of lacquer for 10 to 30 microns. Take a 50 ml beaker and !:ll it with a 1% chloroplatinic acid. Use a No. 18 or 22 gauge wire as a cathode

1G

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17

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onnected (to the ncgative) to a D.C. source of 15 volts. Fhe microelectrode should e connected to an anode (positive) of 15 volts, in series with a 1 Megohm (1 W) :esistor. Pass current through the electrodes for 15 to 30 seconds. Wash the lectrode in distilled water for a few minutes. Use the 1% Na Cl solution with a silver wire, gauge No. 18 to 22, as cathode, and the 15 volts D.C. source in series with the 1 Megohm resistor for depositing a silver-chloride coating on the :ip of the microelectrode. Twenty to 30 seconds will be sufficient. If bubbles come ff from any other part than the tip, it means the insulation is not good and should be redone. Wash the electrode in distilled water for 10 to 15 minutes. Store it in dark container filled with Ringer solution.

The electrode has low resistance (from 100k to 800k depending on the tip), is nonpolarizable and produces very little distortion from D.C. to fairly high frequency (over 1000 Hz) (Figures 10, 11).

In Figures 11A, 11B, 11C, and 11D, the performance of the microelectrode amplifiers is shown.

[PAGE 30]
. A SEARCH FOR AN ELECTRIC FISH ANAESTHETIC THAT WOULD NOT AFFECT THIEIR ELECTRIC SIGNAL

A commonly used anaesthetic for fishes and cold-blooded animals, urethan (ethyl 33
$\mathrm { \ c a r { b a m a t e } = C _ { 3 } \mathrm { F } _ { 7 } \mathrm { N } O _ { 2 } ) }$ has been found to have carcinogenic properties by Wood
and Ball and Cowen"\*. An editorial comment accompanying Wood's report indicated
that the substance named MS-222 (today called Finquel) might be a suitable substitute $\bf \Pi = C _ { 1 0 } F _ { 1 5 } ^ { \alpha } N O _ { 5 } ^ { \alpha S ) }$ was discovered by
for urethan. MS-222 (tricaine methanesulfonate
M. Sandoz during his search for a reliable synthetic substitute for cocaine (benzoil-35
methylecgonine $= \mathsf { C } _ { \underline { { \ P } } \underline { { 1 } } } \mathsf { E } _ { 2 \underline { { 1 } } } \mathtt { N O } _ { \underline { { 4 } } } \rangle$ for a local anaesthetic.
Since Wood's and Ball and Cowen's reports, MS-222 has become routine for anaesthe
tization of fishes to facilitate the handling of both marine and fresh water species. 36
The toxicity of the drug to some species of fish was determined by Markingº. Walker 37 measured its residues in various tissues of salmonides and its
and Schoettger° 38 The chemical and anaes
efficacity has been investigated by Schoettger and Julin 39 , and details about the chemistry
thetic qualities of MS-222 are mentioned by Klontz 40
can be found in the Merck Index

In the last years (1970-71), an agreemcnt has been made betwveen the Sandoz Pharmaceuticals, Division of Sandoz, Inc., Basel, Switzerland, and Hanover, N.J., USA, and the Ayerst Laboratories, New York, N. Y., that thc latter should produce and sell the MS-222 (tricaine methanesulfonate) in the United States under their own brand name of "FINQUEL".

In most of the studies on elcctric fishes, MS-222 has been used as a gencral anaes-41 42 thetic by'the investigators. Szabo\*1, Hagiwara, Szabo and Enger", Enger and Szabo4º, Nobuo Suga\*4, and many others used MS-222 to anaesthetize electric fishes 43 44 during surgical procedures and subsequently to record the electrical activity cither from the electroreccptors or from the nerves conmecting them with the central

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nervous system. No mention has been made of the effects of the MS-222 on the frequency and amplitude of the eleçtrical signals as related to concentration and/or time. 45 mentioned that Hypopomus occidentalis, a South American fresh water weak Bullock
electric fish, with a normal repetition rate range of 25 to 90 per second, would lower its frequency to below 16 per second only under anaesthesia. Under deep anaesthesia, the fish may stop abruptly its electrical activity. By reducing the level of anaesthesia or by stopping it to let the fish recover, it will also abruptly resume the normal electric activity. In our experiments with Sternarchus albifrons, a South American fresh water weak electric fish, we found a gradual tapering of the repetition rate of the electric signal with the deepness of the anaesthesia and a gradual increase of the repetition rate with the recovery. By monitoring the effect of Finquel (MS-222) anaesthetic on the electrical activity of Sternarchus albifrons, it has been observed a fast and significant change in the repetition rate of the electric signals. A decision was made to study the effect of different anaesthetics on the electrical activity of diffcrent species of electric fishes.

The anaesthetic agents of choice were:

1. MS-222 = FINQUEL = TRICAINE METHANESULFONATE

2. NEMBUTAL

3. AMYTAL

4. SECONAL

5. THIOPENTAL SODIUM

6. NOVOCAINE

7. TERTIARY AMYL ALCOHOL

A.INSTRUMENTATION, MATERIALS, AND METHIODS

A specially built 30 em tray made of lucite which could be adjusted to the size of the fish has been used to chcck the cffects of anaestheties on the elcetric organs of elcctrie fish (Figure 12). The tray is provided with fittings for the rapid discharge of

25

[PAGE 32]
anapsthcties or water and a constant aeration of thc solution is possible if nccessary. It also has at every centimeter distance embedded stainless steel elcctrodes connected on both sides of the tray with the exterior and to contacts. The electrodes corresponding to the position of the head and tail of the fish were connected through a high-gain, low noise amplifier to two oscilloscopes Tektronix (one for photo-taking), a counter and an FM tape recorder (Figure 13). The tray with the fish was located in a floating screen room and all the instrumentation grounded to the screen room was located in the laboratory outside the screened room. A lucite cover on the tray prevented the fish from jumping out, different holes served to pour in water or anaesthetics, to put in thermometers,and to let excess air out. A d-c (battery) operated high intensity lamp was used to illuminate from the top the fish and to furnish enough heat to hold the temperature constant during experiments. For the experiments, the fishes' own aquarium-water was used at the beginning of the experiment and the same water was used for mixing in the anaesthetics. In this way, the temperature of the water and anaesthetic solution was easy to keep to the same level as the normal temperature of the water in the aquarium. The pH of the aquarium water at the beginning of the experiments and also the pH of the anaesthetic solution were measured with an expanded precision type of pH meter. The pH meter has been calibrated before each experiment.

Tentative measurements and observation of the action of the anaesthetic have been made first on goldfish and then on onc of the electric fishes before another specimen has been selected for the experiment. For each experiment with one and the same anaesthetic, five specimens of Sternarchus albifrons have been uscd. The specimens of Sternarchus albifrons, a fresh water South American sternarchid weak electric fish, have been in our laboratory for over one year and they were all healthy and varied in weight from 14 to 30 grams. Gymnarchus niloticus, a fresh water African gymnarchid weak clcctric fish,' has been kept for over six months in our laboratory. There is another specimen just reccived. There are also two Gnathonemus petcrsii, fresh water African mormyrid eleetrie fish, being in the laboratory for over two months.

26

[PAGE 33]
27

[PAGE 34]
The fishes have been weighed before each experiment and put in the experimental tray in their own aquarium water. The electrical activity of the electric organ has been monitored, recorded with the FM magnetic tape recorder, its amplitude measured on the calibrated oscilloscope, and a photo taken. After a few minutes, the water was discarded from the experimental tray and the anaesthetic was introduced with a funnel through one of the holes in the tray cover. The time of introducing the anaesthetic has been marked with the aid of a timer and also recorded on the magnetic tape and in our records. The effect of the anaesthetic on the fish, its behavior and respiration were constantly observed. The electric activity has been monitored and from time to time a photo has been taken from the oscilloscope. The moment in which the fish was anaesthetized completely has been recorded. The fish respiration and electric activity (amplitude, wave form and repetition rate) were constantly observed. If necessary, the anaesthetic has been immediately discarded and fresh aquarium water has been introduced in the experimental tray with adequate aeration for the fish. This moment has been recorded and the recovery time of the fish has been marked. The electric activity also has been monitored. When the fish was considered completely recovered, it was returned to its own aquarium. Sometimes because of the long time a fish was anaesthetized, it has been returned from the experimental tray in a net floating in its own aquarium with adequate aeration with bubble stones under the net.

Experiments were performed on five specimens of Sternarchus albifrons (No. 2, 3,4, 6 and 7). In order to assess the effectiveness and dosage of the "MS 222 Finquel" tricaine methanesulfonate, we rccorded the electrical activity before, during, and after anaesthesia. The frequency of the clectric organ will drop immediately after adding the tricaine methanesulfonate to the water in a special tray,provided with stainless steel elcctrodes sct at a distance of one cm from one another over the length of tho lucite tray. The fish were restricted by a partition and a "U" shaped lucite device placed on top of the fish. Figure 14 shows the decrease in frequency

28

[PAGE 36]
with time when the fish has been kept in a 1:10000 MS222 anaesthesia solution in water and the return of the impulse rate after the solution has been exchanged with fresh water. The electrical activity of the elcctric transmitting organ was recorded on a Hewlett-Packard four-channel FM magnetic tape recorder to be played back and analyzed later. Figure 15 shows the electric activity before anaesthesia; Figures 16, 17, 18 and 19 show the electric activity during anesthesia; and Figures 20 and 21 show activity during the recuperating process.

B. RESULTS AND DISCUSSION

From seven anaesthetic agents we tried, six were inadequate because the repetition rate of the electric signal was affected. Only the thiopental sodium Abbot (pentothal sodium) does not influence the electric activity of Sternarchus alhifrons.

The thiopental sodium has been checked on five different fishes (same species). It has a very fast effect in a dosage of 1:10000 in water. The fish, after it has been anaesthetized, would remain as such for 2 to 4 hours lying very quietly. It recovers completely and the anaesthetic has no ill effect on the fish. We anaesthetized themn repeatedly and after six months' time they are doing well. After the fish has been anaesthetized, it has to be put in aerated fresh water where it can remain for hours. Figures 23, 24, 25 and 26 show that the repetition rate of the electric signal has not been affected in over 40 minutes. The only change was produced by a slight deerease in water temperature (from 720 pulses to 700 pulses) of about 0.4 C. Sternarchus albifrons is very sensitive to changes in water temperature. It will inerease the repetition rate of the elcctric signal for an increase in water temperature and it will decrease the repctition rate for a decrease in water tempcrature. For every degree Centigrade, it may change the repctition rate by about 50 to 80, depending on the particular fish.

Tluopental sodium or Sodium 5-cthyl-5-(1-methylbutyl)-2-thiobarbiturate has the chemical formula $\mathfrak { c } _ { \mathfrak { L } \mathfrak { L } _ { 1 7 } } \mathfrak { N a N } _ { 2 } \mathfrak { O } _ { 2 } \mathfrak { S }$ , a molccular wcight of 204.33 and is a yellowish-

[PAGE 41]
white, hygroscopic power, soluble in water and alcohol and is a strongly alkaline 46 solution. The Abbot preparation is of nonhygroscopic crystals

Tubocurarine chloride can be administered by intra-abdominal injcction. There is no interference between tubocurarine and thiopental sodium.

The stability of thiopental solutions depends upon several factors, including the diluent and conditions of storage. It is recommended to keep them under refrigeration and tightly stoppéred.

"Finquel" or "MS222" is a meta-amino-benzoic-acid-ethyl-ester in the form of tricaine methane-sulfonate and has the chemical formula $\mathsf { C } _ { \mathsf { 1 0 } } \mathsf { \Pi } _ { \mathsf { 1 5 } } ^ { \mathsf { H } } \mathsf { N O } _ { \mathsf { 5 } } \mathsf { S }$ with a molecular weight of 261.31 and is produced as fine needles, soluble in water. It is slightly acid and is stable to boiling.

Finquel 1:10000 in aquarium water would affect the repetition rate of Sternarchus albifrons and make it decrease in 10 minutes from 780 to 440 (Figures 27 and 28).

35

[PAGE 43]
Four specimens of Sternarchus albifronshave been used for establishing mcthods to record the autorhythmic activity of their electrorcceptors (Figures 29, 30). The fishes have to be anaesthetized and curarized in order to avoid twitching of the muscles during microclectrode recordings. We tried the effects of d-tubocurarine on
42 Sternarchus albifrons.. Hagiwara, et al", recommended 0.05 to 0.1 mg curare/fish
43 and Enger and Szabo°recommended 0.03 mg d-tubocurarine/g fresh fish weight. Both used (MS 222) tricaine methancsulfonate for anaesthesia (1:150, 000). We foud that the quantities given did not correspond in our case.

The weight of the Sternarchus specimens varied between 16 g and 19 g. One specimen (16 g) received 0.5 mg d-tubocurarine intra-abdonminally. The fish was paralyzed in one minute. Electrical activity of the main electric organ subsided after 20 minutes and the fish was dead after another ten minutes. All the time the fish was kept in a 4000 ml beaker with medicated aquarium water at a pH of 7.0 and a temperature of 23,0°C. An aerator stone provided the necessary air.

A second specimen of Sternarchus (also 16 g) has been anaesthetized with MS 222, 1:25,000. One gram of MS 222 has been dissolved in 1000 ml distilled H0 as stock solution. Then this was diluated to the proper amount in a 4000 ml beaker by adding aquarium water with merbromine and acriflavine added as disinfectants. The MS 222 dilution of 1:150, 000 would not affect the fish in over one hour. The dilution of the MS 222 was reduced to 1:25,000. After 20 minutes in the MS 222 solution the fish was injccted intra-abdominally with 0.05 mg of d-tubocurarine 1 mil solution. The fish was paralyzed in thrce minutes. For one hour it gave a good strong elcctric signal of the maių clectric organ, after. this gradually it diminished in strength and in another hour the fish was dead.

IV. TECHNICAL DISCUSSION

[PAGE 45]
y the data given by the previous authors did not correspond for our

nens.
third specimen of Sternarchus (18 g) was first anaesthetizcd in MS 222
,500 for 20 minutes after this it was injected intra-abdominally with 0.1 ml
.05 mg/ml d-tubocurarine (Figure 31). The fish has been placed then in a
cial recording tray with circulating aerated water with merbromine, acri
vine and MS 222, 1:75,000. Recording from the electroreceptors has been
tempted with a rieutral electrode in the tray water and a silver-silver chloride
nicroelectrode placed on a tonic, ampullary electroreceptor (Figure 32). The
strong electric signal from the main transmitting electric organ masked the weak
sutorhythmic activity of the electroreceptors. This specimen has been returned to its tank after 30 minutes and recovered almost instantly and is doing well since then. its tank after 30 minutes anu 1 received the same treatment as the water,2
Another fourth specimen of Sternarchus (19 g) receivcd the same treatment as the
third specimen. This time instead of using the neutral electrode in the tray water, a
silver-silver chloride wire loop 1/4 in. diameter was placed around the tonic, ampullary electroreceptor (Figure 33). This time the main electric organ signal did not mask the recordings of the authorhythmic activity of the electroreceptor. After 40 minutes, the fish was returned to its tank and recovered almost instantly, like the previous specimen, and is doing fine without any ill effects since then (Figure ile w r …from
34).
The microelectrode recording experiments were performed in a shiclded room from shich all a-c instrumnents, plugs and cords were eliminated and the shiclded room then constituted a floating ground. Mlumination of the specimen was done by a microseope Iamp fixed on the stereo-microscope and connccted to a 6 volt car battery. All keaters were taken out of tho water and all amplificrs were connected to batteries. Cher equipment, such as the microscope support, the microscope, the jack, the

[PAGE 46]
Figure 31. Sternarchus albifrons specimen after d-tubocurarine injection.

Figure 32. Ampullary, tonic electroreceptors of Sternarchus albifrons. The white points are ampullary receptors. The dark points are tuherous, phasic reeeptors.

40

[PAGE 47]
Figure 33, Set-up for recording autorhythmic activity of ampullary electrorcceptors of Sternarchus albifrons, The silversilver-chloride wire loop neutral electrode is visible.

Figure 34. Anacsthetized,curarized Stcrnarchus albifrons in the recording {ray. Carbon electrodes record the activity of the transmitting organ as seen on the oscilloscope.

41

[PAGE 48]
plifiers, etc., was connected to one point ground and to. the Tektronix T22 modified fferential amplifier ground. The microolectrode amplifier and support was changed om the integrated circuit model used bofore to an electrometer tube type and a three ransistor amplifier which is less sensitive to the change in impedance produced by light movements of the fish than the previous one. The microelectrode, a silversilver-chloride, platinum, silver-chloride electrode of 0.5 micron tip diameter, was positioned on the electric receptor inside the grounding loop. Figure 22 shows the recording setup and Figure 35 the schematic of the microelcctrode amplifier.

At the same time, the electric activity of the receptors was displayed on one of the traces of a dual scope. The activity of the electric transmitting organ was recorded with two carbon electrodes placed at the end of the tray and commected to another Tektronix T22 amplifier and displayed as a second trace on the scope. A Hewiett-Pàckard four channel FM tape recorder was used to record the electrical activity of the electric receptors on a magnetic tape. It can be played back and analyzed at a later time. Photos were made during the recording. Figure 37 shows the activity of a synchronous tonic electroreceptor (in a previous report we mentioned that we re- . corded from a nonsynchronous tonic electroreceptor), and Figure 38 shows the electrie activity of a nonsynchronous phasic electroreceptor. After finishing the experiment, the fish was injected with neostigmine methylsulfate $1 : 1 0 ^ { 6 }$ to counteract the effect of the D-tubocurarine. The fishes recuperated in a few minutes and are doing well.

Another experiment was performed with two Gymnarchus niloticus,Cuv. Each of these fishes was placcd in 25 gallon wator tanks with lueite trays raised to approximately 4 inches from the top. Gymnarchus niloticus is an air breather and if left in a deep tank must expend excessive energy to swim to the surface for brcathing. The tank is kept clean by lucite plates provided with holes to Ict dirt fall to the bottom where it can be vacuum-clcaned very casily through special holes. The tánks of

[PAGE 50]
3. High impedance microelectrode de follower preamplifier.

Fig. 37. Sternarchus albifrons 6. Synchronous tonic electroreceptor. Microelectrode recording, 1 msec/em, 100 mV/cm.

38. Sternarchus albifrons 7. Nonsyn-'mous phasic clcetroreceptor. Micro-:trode recording, 2 mscc/em, 100 mV/cm.

Fig. 39. Gymnarchs niloticus Cuv. #2. Normal electric activity of the transmitting electric organ. Not comnccted with the tank of another Gymnarehs. 2 msec/cm; 1 mV/cm.

44

[PAGE 51]
nnarchus niloticus are approximately thrce feet apart. .The covers and outside magnctic stainless steel frames were all grounded and so was the tark through bon electrodes placed in the filters.

¿o carbon electrodes in lucite tubes (with holes) were placed at a distance of 15 cm m each other alongside the fishes. A third electrode was placed either midway tween the recording electrodes or on one of the ends of these electrodes. The first o electrodes for cach fish were connected with the T22 Tektronix amplifiers and nnected to two different Tektronix scopes. The third electrodes were connected a DPDT switch which could actuate also a battery to raise the lower trace of the cove when the two electrodes in the two tanks were connected and hence a communicalon link was established between the two Gymnarchus to study the effect of social nteraction and communication between them.

n Figure 39, the normal electrical activity of the electric organs of Gymnarchus lo. 2 and in Figure 40 the electrical activity of Gymnarchus No. 3 can be seen. 'igures 41 and 42 show that Gymnarchus No. 2 almost stopped the electrical activity r ten to twenty seconds.Figures 43and 44 show the modified activity of the imlses from Gymnárchus No. 3, and Figure 45 shows the modified activity of Gymnarchus o. 2. When a metal rod was introduced in the tank of Gymnarchus No.3, Gymnarchus . 2 would nervously move back and forth and eventually attack the electrode connected th the other fish. This is only the beginning of studying the communication between o Gymnarchus niloticus.

e Gymnarchus niloticus baby was sectioncd and fixed in buffered formaldehyde (10%) about one month. Thcn it was decalcified with Kristensen's decalcifying solutions 24 hours. After this, it was dchydrated using cthyl alcohol, toluene, tolucne with affin, and finally embedded in degassed parafüin with 56°C melting point.

[PAGE 52]
0. Gymnarchus niloticus Cuv. #3. Normal tric activity of the transmitting electric mn. Not connccted with the tank of another nnarchus. 2 msec/cm; 1 mV cm.

12. Gymmarchus niloticus Cuv. 2. Elce-: activity of the cleetric organ when fhe ': has been clectrically connected through bon clectrodes and a wire and switch with tank of another Gymnarchus. 2 msce/em; V/cm.

Fig. 41. Gymnarchus niloticus Cuv. #2. Electric activityof the electric organ when tank has been electrically connected through cąrbon electrodes and a wire and switch with the tank of another Gymnnarchus. 2 mscc/cm; 1 mV/cm.

Fig. 43. Gymnarchus niloticus Cuv. 13. Electric activity of the clectric organ when the tank has been clcetrically connccted tlurough carbon electxles and a wire and switch with the tank of another Gymnarehus. 2 mscc/em: 1 mV/cm.

[PAGE 53]
Malapterurus elcctricus, the clcctric catfish (Figure 51) is different from the other lectric fishes because it does not obey Pacini's law. All tho othcr electric fishes bey Pacini's law, according to which the inncrvatcd faces of the electroplates be-47 ome negative during the dischurge, whatever the anatomical orientation of the organ. his fact is due to thc unique anatomy of Malapterurus. The electric organ of alaptcrurus forms a sort of loose jacket around the fish, instcad of being embedded

[PAGE 56]
3

r    tec. m les d ne

C

[PAGE 57]
thin the body muscle as in all the other elcctric fishes. The clectric organ, depend-; on the fish's sizc, forms a sheath from 2 to 10 mm or even thicker, in which the ctroplates are stacked in the longitudinal dircction arranged in a somehow nonlerly fashion, contrary to the othcr elcctric fishes. The electroplates are disks about 1 mm diameter and 20 to 40 microns thickness and derived from myeloblasts. the center of the caudal face of each elcctroplate, a thin stalk arises from a complex agination. The end of this stalk makes contact with the branches of a single myelinated ve fiber arising from a large ncrve cell situated in the gray matter of the spinal cord ween the levels of the first and second spinal nerve roots. As it can be seen on ure 51, the nerve trunk which is heavily myelinated is easily identified. The way vhich the electric organ is innervated plays an important role in the synchronization chanism of the discharge.

Malapteruridae discharge consists of a long train of 15 to 30 monophasic pulses to 2 msec duration in a total time-interval of approximately 100 milliseconds.

x to seven inch electric catfish couid discharge impulses of approximately 200 V. ny surprise, Dr. Earl Harold from the California Academy of Sciences and Stein-It Aquarium in San Francisco, told me that the two very large (approxim tely 2 to ot) electric catfishes which the aquarium had did not discharge more than 100 volt es.

surprising that theMalapteruridac we checked could put about 0.5 w/hr of energy ach gram of elcctrić tissue of the electric organ. It is our intontion to investigate fact more closcly. Tho electric dischargc of three strong fresh water electric sMalapterurus clectricus weighing between 55-65 g was measured when in the r and out of water in the special tray with contacts. They discharged between )0 volts in water and between 150-190 volts out of water. The discharge consists rsts of 5 to 7 impulses of 1:5 to 2 msec duration (Figures 52, 53, 54).

[PAGE 59]
53

[PAGE 60]
In our synoptic Table No. 1, thc past achievcments and results of the study are mentioned, the proposed continuation work is shown, and the future objectives are delineated.

The multimodulation-multicoding communication system of elcctric fishes served as a nodel in the development of a communication technique to resist jamming.

'he physical analogs of a phasic and a tonic electroreceptor have been established for re Sternarchus albifrons, a South American fresh water weak electric fish of the high equency type (Figures 55, 56). The electroreceptors of other species have to be vestigated and the relationships of the various electroreceptors to tho underwater ttern recognition systemwill be established.

mentioned the effect ol moving objects past the electroreceptors in.onc or another :ection. Some of them would respond in one way for, the forward direct (head tó tail) increasing the rate of the impulses and in another way for the backward direction

[PAGE 62]
current and voltage $\mathbf { r _ { g } }$ generator internal resistance

external resistance $\mathsf { G } _ { \mathtt { a } }$ autorhythmic generator

external canal resistance ${ \pmb { \mathrm { r } } } _ { 1 } { \pmb { \mathrm { \cdot } } } _ { 1 } ^ { \bullet } { \pmb { \mathrm { r } } } _ { \mathbf { i } _ { 2 } }$ internal resistance

external canal capacitance $\mathbf { c _ { s } }$ internal receptor capacitance

receptor inner face resistanco ${ \varepsilon } _ { \mathfrak { s } }$ skin resistance

[PAGE 64]
(tail to head) by decreasing the rate of impulses. Other electroreceptors will rcact to higher or lower conductivity than the medium (aquarium water) in a similar way. Obviously, the rosponse of the electrorcceptorș in othcr electric fish specics have to be assessed in order to get a better overall picture of the underwater pattern recognition process. Figures 57 through 63 show two different electroreceptors of electric fish. The results obtained from the electric receptors study will lead to a simulation plan of an underwater paitern recognition system. A passive hybrid underwater pattern recognition system block diagram is shown in Figure 64. In this block diagram beside the electric sensory receptors, the other types of lateralis line sensory receptors are combined to obtain a better cross-correlation of the different signals. Eventually passive optical sensory receptors will be added (for detecting, for example, changes in the bioluminescent organisms in the water when disturbed by a stimulus like heat, water turbulence, etc.).

With regard to the other lateralis line organs, Sternarchus albifrons, like other teleosts, have canal organs as well as superficial organs, called free neuromasts. Some particularities of these organs should be noted. Not all canal organs are in direct relation with the external environment. The sense organs do not always alternate with the pores 48 of the canal °. In some cases, three sense organs are located between two pores. All three cupulae lie in a "wątery" fluid filling the canal. The fluid seems to be a form of olymucosucharides. The canal is not free from one pore to another, but is obstructed etween the front and second, and the second and third cupula-sense organ units by two :pithelial plugs. Thus, the middle unit is not in direct continuity with the external enironment (Ref. 23). It should be mentioned that Gymnarchus as well as Notopterus ave a completely closed canal system. The free ncuromasts found in the head of ternarchus are relatively large, are protected by paired prominent epidermal flaps nd have a histological structure similar to 'big pit organs". As in the pit organs of ymnarchus (Ref. 25), these free organs of Sternarchus are innervated by a bundle

[PAGE 65]
59

[PAGE 66]
cn

[PAGE 67]
59. Ampullary tonic electroreceptor. (Schematic)

Fig. . 60 Stimulus and recording from an ampullary tonic electroreceptor.

61 Tuberous phasic electroreceptor. (Schematic)

Fig. 62 Stimulus and recording from a tuberous phasic elcctroreceptor.

G1

[PAGE 68]
62

[PAGE 69]
IIGGUE

PASS     EM

63

[PAGE 70]
ick and thin fibers. The specialized lateral line organs (elcctric scnsory organs) be subdivided into two main groups: ampullary organs and tuberous organs. :e are many subdivisions of these organs and we have previously reported about 1.

different lateral line organ types, canal organs, frce ncuromasts, ampullary ns and tuberous organs, have a characteristic distribution pattern being similar ymnotids, except for Electrophorus electricus. The density of each type of organ 3 same species depends upon the size of the specimen, e.g., canal organs in the region are separated by 1 mm in a Sternarchus albifrons 13 cm long, and by 2 mm e 22 cm long.

pproximate distribution of single organs was established by observing the "pore rn" on the surface of the skin with a dissecting microscope. It follows from the ous descriptions of these organs that these "pores" should not be considered as 1 hole in the integument in every case, but rather a local differentiation of the rmis overlying the sensory organs.

[PAGE 71]
V. RESULTS AND CONCLUSIONS

e was to devclop the necessary instrumentation for microclectrode recordings etroreceptors of electric fish so as to be able to investigate their pattern ion ability. In the previous chapter we demonstrated that the system is workand we succceded in recording the autonomous autorhythmic electrical activity nic ampullary electroreceptors of Sternarchus albifrons compared with the organ's normal activity (Figures 65 and 66).

ording was made during resting of the fish with no stimulus. The impulses egular around a repetition rate of between 100 and 300. The amplitude was :. 5 mv per spike. The spike duration was very short - around 200 micro-(Figures 65, 66).

r investigation, it can be concluded that electric fishes could use their elecins (transmitting and receiving) for navigation and communication - in other attern recognition. 一

ordings and histological evidence show that Sternarchus albifrons has three electroreceptors: ampullary tonic nonsynchronous units, ampullary tonic ious units, and tuberous phasic nonsynchronous units. The physical analogs ind phasic electroreceptors are shown in Figures 57 and 58. Both are reprev a generator connected to resistancos and capacitances in series and in

The difference between tonic and phasic electroreceptors is that the first e one resistance in sceries with the generator whereas the phasic elcctros have a capacitance. The tonic clectroreceptors secm to be predominant, e five-to-one, compared to the phasic electroreceptors. The clectroreseem to act, to a certain extent, independently of the main elcctric transmitting .least two out of three different tpes of clectrorcceptors are asymchronous

[PAGE 72]
66

[PAGE 73]
ne type of electroroceptor will synchronize with the main electric organ. ound that the complete denervation of the transmitting elcctric organ does e activity of the asynchronous electroreceptors (both phasic and tonic). The 1 capable of responding to conductive and nonconductive objccts placed near xody. It may affect the total capability in determining certain movoments or a certain extent, its sensitivity in pattern recognition. Some of the synnic units are connected to one and the same nerve trunk part of the acoustico-'stem but connected to specialized big nuclei in the brain.

triking fact about fresh water weak electric fish, besides their spontaneous gan, is that all of them are provided with a highly developed lateralis line elated to this acoustico-lateralis system is an enlargement of the cereecially in Gymnarchus niloticus and in mormyrids. The unusual importance ilis system in these fish, compared with other teleosts, is not due to an innber of "ordinary" lateral line sensory organs, but rather to the existence umber of specialized sensory organs within this same system.

xorting our hypothesis about a hybrid complex underwater pattern recognition l by electric fishes in recognition of prey, predators, and navigation in is recommended that the other lateralis line systems from different fresh electric fishes should be studied with the aim to find out the role of the isory organs in pattern recognition.

: of the electric fish pattern rccognition system would make it possible to models of the physical analogs of the sensors could be integrated in object location, dctection and identification. The range and scnsitivity of the i be assessed and improvements could be made.

lely used anacsthetic,- "MS 222" or "Finqucl" (tricaine mcthanesulfonate) - cts the repetition rate of the electric impulses of the clcctric organ. It o do a series of experiments on differcnt anaesthetics to establish whcther

[PAGE 74]
¿e is one which would not affect the frequency of the impulses. It has been found : thiopental sodium (sodium penthotal) will not affect the frequency of the impulses is a safe anaesthetic for fish, acting fast and without any ill effects.

er subdivisions exist between the one and the same type of electroreceptor, but this not been as yet investigated in a detailed way. The connections between the electroeptors, the different nerves, nerve-trunks and the brain have to be investigated. In : way, their interrelationship could be established. Microelectrode recordings from electroreceivers proper and from their nerve fibers have been planned. Electrosection and clearing of the lateral line near the electroreceptors to be investigated . enable us to record from the efferent nerve fibers.

m the experiments with Gymnarchus niloticus, we concluded that one fish would ognize and communicate with another one of the same species. Behavioral experiits in this direction will be continued. It is recommended that further microetrode recordings be made from the clectroreceptors and from the nerve fibers of marchus albifrons and of the newly received Gymnarchus niloticus.

; possible to simulate an equivalent sensory system responding to different stímuli erwater. A system with a double feedback mechanism can be envisaged: (1) one resented by a constant frequency electric field transmitting system operating on phase-synchronous electroreceptors responding to discontinuities in the electric d or to changes in the phase relationship transmitter-receptor; and (2) another represented by a variable frequency transmitting system responding to disturbances he field between transmittcr and receptor with a change of the frequency of the ismitting elcctric organ. To this we could add an independent dual autorhythmic eptor system: (a) responding with the increase or decrease of the autorhythmic juency depending on movement direction of the disturbance in the electric field; (b) rosponding with a change in the latency depending on the magnitude of the disxnce, also distinguishing between conductive and nonconductive objccts.

[PAGE 75]
mnarchus niloticus, one of the most sensitive electric fishes, served as a modcl for simulation of a simplc pattern recognition system (Figures 67 and 68).

[PAGE 76]
70

[PAGE 77]
d   e dps)(

71

[PAGE 79]
VI. REFERENCES

[PAGE 82]
76