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ELECTRIC FISII INVESTIGATION

Final Report

Contract

8 March 1973

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ILLL:TRATIONS

Figure Page
1 Mauthner cells 4
6
2 Electrorcceptors
3 Cranial nerves of Electphorus 8
4 Microelcctrode amplifier 13
5 Microelcctrode amplifier: schematic diagram 13
6 Loss in -dB with incrcasing 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 or measuring microelectrode resistance 19
20
11A Amplification Lactor, square wave
11B Amplification factor, sinusoidal wave 21
22
11C Recordings at different square wave
11D Recordings at different square wave 23
12 Adjustable Lucite Tray 27
13 Instrum.entation for Anacsthesia 27
14 Solution 1:10000 in water on Stcrnarchus albifrons 29
15 Sternarchus albifrons #7 normal elcctrical activity 31
16 Sternarchus albifrons #7 in ancsthetic tricaine methane 31
sullonate, 1 minute
17 Sternarchus albifrons #7 in anesthctic tricaiie mcthane 31
sulfonate, 2 minutes
18 Sternarchus albifrons f7 in anesthetc tricnine methane 31
sulfonate, 4 minutcs
19. Sternarchus albifrons 7 in anesthetie tricaine metiane 32
sulfonate, 5 minutes
20. Stcrnarchus albifrons recuperating. 5 minutes 32

U

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二二 二
21 Stcrnarchus albifrons recuperating, 8 minutes 32
22 Sct-up for microelcctrode 32
23 Sternarchus albifrons No. 1 just before anacsthesia 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.)
26 Sternarchus albifrons No. 1 anaesthetized with thiopental sodium 34
(37.5 min.)
37 Sternarchus albifrons No. 2 in aquarium water 36
28 Sternarchus aIbifrons No. 2 in MS-222, 10 minutes 36
29 Set-up for recording 38
30 Close-up of the set-up for recording; 38
31 Sternarchus alhifrons after d-tubocurarine injection 40
32 Ampullary, tonic electroreceptors 40
33 Set-up for recording 41
3.1 Anaesthetized, curarized Sternarchus albifzons 41
35 Microelcctrode amplifier 43
3G Preamplifier 44
37 Synchronous tonic electroreceptor recording 44
3S Nonsynchronous phasic elcctroreceptor recording 44
39 Gymnarchus niloticus Cuv. #2 electric activity 44
10 Gymnarchus niloticus Cuv. #3 electrio activity 46
41 Gymnárchus niloticus Cuv. #2 electric activity 46
2 Gymnarchus niloticus Cuv. #2 electric activity 46
13 Gymunarchus nfloticus Cuv. #3 electric activity 46
44. Gymnarclus niloticus Cuv. #3 electric activity 47
5 Livn narchus niloticus Cuv.#3 clcctric activity 47
il. Gymnarchus niloticus, Cuv. (baby) - trinsversal cut 48

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Figure Pago
47 Gymnarchus niloticus Cuv. - air bladder and spinal cord 49
z
48. Gymnarchus niloticus Cuv. - spinal cord 49
49 Gymnarchus niloticus Cuv. - brain 49
50 Delafield - Harris hematoxylin staining method 50
51 Malapterurus electricus 52
52 Malapterus elcctricus on the scale 53
53 Device for measuring the voltage 53
54 Discharges of the electric rgan of Malapterurus electricus 54
55 Tonic, ampullary electroreceptor 56
56 Phasic, tuberous electroreceptor 57
57 Tonic electroreceptor 59
58 Phasic electroreceptor 50
59 Ampullary tonic electroreceptor 61
60 Stimulus recording 61
61 Tuberous phasic electroreccptor 61
62 Stimulus recording 61
63 Electrorcceptors of Gymnarchus niloticus 62
64 Underwater pattorn recognition system 63
65 Recording from an anaesthetized, curarized 66
Sternarchus albifrons specimen
66 Microelcctrode recording 66
67 Gymuarchus niloticus- photo 70
G8 Gymnarchus niloticus - placcincnt of elcctric 71
transmitling and rccciving organs
69 Fish Laboratory A 72
70 Fish Laboratory B 72
Table
1 Undkrwater patlcrn recognition system 55

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SUMMARY

The elcctric organs of Stornarchus albifrons, a South Amcrican fresh water weak elcctric fish, have been stulicd with emphasis on clectroreceptors. The morphological aud physiologicai characteristics of clectroreccptors, ampullary and tuberous, a. e discussed. Spccial instrumcntation required for the role of these elcctroreceptors in pattern recognition has becn devcloped.

We have recorded with microclectrodes tne autonomous autorhythmic electrical activity of the tonic asymchronous ampullary electrorcccptors of the South Americaa weak fresh water electric fish Sternarchus albifrons. We have also recorded the electrical activity from the phasic tuberous electroreceptors and of the synchronous ampullary electroreceptors of the same electric fish, Sternarchus albirons. Preliminary mcasurements 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 somo specimens of the African weak fresh water e ectric fish Gymnarchus niloticus. They are supposed to be the most sonsitive 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 Saprolegnia fungus and could not be saved, but we fixed a number of them in buffered formaldehydle and one of them has been cut and mounted in paraffin for histological studics of the elcctric organs. Preliminary measurements have been made ou the communication capability of adult Gymnarchus niloticus.

A study of the anesthctizing cffect of tricaine-mcthanesulfonatc (MS222 = FINQUEL) on Sternarchus albifrons has beon undertaken by plotting time for the ancsthesia and recovery for different specimens.

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A study of an anaesthctic which docs not affect Uhc elcctric fish's elcctric organ pulse repctition rate is presented.

Also, the eflect of D-tbocurarinc and the counter-cffect of ncostigmine has been assesscd for Sternarchus-albifrons. Finally, some improvemcnts in the microelectrode recording instrumentation have been nade.

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

The other lateralis line system sensory receptors, like mechanical receptors and displacemcnt receptors, have been discussed as part of a general hybrid pattern recognition system of the flsh. 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 reccivers and electric fields. More histological work 1s needed to establish the r:.lationship 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 cur prcvious rcports, we described the morphology of the electric organ 1 of Stcrnarchus albifrons, a weak fresh water electric fish from South America. We menticned that the electric transmitting organ of Stcinarchus 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 morlulated.

The form, rate, and amplitude of the signals emitted by the electric fishes are as diverso as the forms and sizes of these fishes. Some of the weak signals are used to locate cbjects or animals in their environmient, or for navigation, species recog-2
nition, and communication. The strong electric discharges servc for offense or 3
defense. Watanabe and Takeda" investigated the effect of a-c current with a frequcncy clcse to thc eleci Ical signal emitted by Eigenmannia. When the applied pulses came within + 3 to 4 pps of the one emitte by Eigenmannia, the fish would change its rate by 4 to 5 pps in a dir ction which increasori the pps sepaiation. Increasing the frequency of the appiied  curront In 1 pp.crements caused tho fish to shift its frequcncy 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 expcriments with Sternarchus albifrons, but thc applied a-c signal was within 0.5 cycle of the signal of the Lsh, demonstrating how spccific the applied signal must be to elicit a change in the Sternarchus signal rate: + 0.5 cyclc is the range of selcctivity of the fishes' clectrical transmitting-rccciving system. The fact that many fresh water and sea water clcctric fishes have never beon studicd may present some difüculties in obtaining spccial kinds of clectric fishes, and thcir carc may not bo an easy task. There are, however, enough species to enable many experiments.

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Very little is known about thc clcctrical activity of marine clectric fishes except Torpcdo and some rays. Narcine Barziliensis is the only known marine electric 4
fish having twvo dlifforent clectric organs. Bennett" studied the mode of oporation of the eloctric organs of Raja eglantaria, a marine elcetric fish, and of the fresh water fishes H:popomus and Stcrnopygus and compared them with Narcine, Mormyrus, Steatogenys, Gymnorhamphychtis, Malapterurus, Gymnotus carapo, and Electrophorus electricus.

The main objects of this study were the form, innervation, and physiology of the electroplates forming the electric transmitting orgars. The clectroplates 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, Malaptcrurus, and most of the Mormpridae, 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 becn compared for elcctric fishes like
Sternopygus (rate = 50/scc) and Eigcnmannia (rate = 280/sec) with the Sternarchidac
(max. 1500/sec). Compared with mammalian central nervous systems, pcak fre
quencies of the elcctric organs are not greatly diffcrent. The Renshaw cell can 5
discharge impulses at a rate of 1400/sec, :nd neurons in the sensory pathi some-6
uimes produce bursts at a rate of about 1000/scc.

In an examination of discharge pattern and organ function studics, it was noted that Grundest? defined two groups of clectric fishes: those that cmit signals at a constant

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rate, and those that emit pulses with a variable rato. For examplc, Gymnarchus, Sternopygus, and Eigenmannia are in thc constant rate group; Elcctrophorus, Gpthonemus petersii, Stcatogenys, and Hypopomus belong to the variable rate group. Bennett\* did not make any conncction between +ho electric fishcs' electrical 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 breedingof 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 se m to be related to informatia regarding movement of objects near or around t' sh.

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 pectoral 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 roquirement of high speed
f transmission in synchronized systems like many of the elcctric transmitting
organs. This has been useful in predicting sites where transmission has been

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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 Vill-th nerve conncctions und collaterals deriving from the excited Mauthmner ccll are shown).

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

Th, neural systems controlling electric organs havc provided a largc number of exi.mples of electrically mediated transmission, which meets the functional require ment 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 corrclation 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 general interest. Some knowledge is being obtained of afferent pathways from electroreceptors in weakly electric fish which have inportant inputs to the electric organ control system. Both operant and respondent conditioning of the control system can bè obtained and conditioned response latency can be very short. It is not unreasonable that the complete neural pathway of the conditioned response could be cbtcined in these cases. The central connections are still minimally explored; one kuows 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.

In Stemarchus albifrons clcctrorcceptors are distrikuted over the entire body. The rhasíc tuberous receptors are very numerous as compared with the tonie ampullary receptors (Figure 2). The deusity of receptors is greatest in the head region and

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Tonic, Ampullary Receptor

Phasic, Tuberous Receptor

Figure 2. Anatomical diagrams and equivalent circuits of electroreceptors in fresh water clectrie fish: (a) tonie, ampullary receptor an? (b) phasie, Auberous receptor. Diagrams are shown with the exíernal mediuin to 'the top. The skin and wall of receptor cavilies are shown in cross scetion as lines. The opening to the exterior of phasie receptor cavity is shown as occluded by a porous mass, The nerve fibers innervating receptor ells are indicated,

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

In Figure 2 the equivalent circuits of tonic, ampullary and phasic, tuberous organs is shown. Cross sections through the reccptors are shown with the external mediun 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 rums 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 c' .ically mediated 20 transmissions at tonic receptors of gymnotids.Tie morphologicai characteristics of the snyapse are those typical of chemically mediated transmission. A strong brief anodal stimulus produces an evoked response Icng outlasting the stimulus. Then there is & synaptic delay between the initiation of the impulse and the nerve impulse (between 0.5 and 1.5 msec). Mormvrid tonic receptors are similar to those of gymnotids and Gymnarchus niloticus tonic receptors are morphologically similar,

The relationship betwecn the dilfcrent elcctroreceptors of Sternarchus albifrons in pattern recognition has not as yet been studicd. The present study developed special instrumentation required for investigaiing the roles of these electroreceptors in pattern recognition and obtained prcliminary measurements of electrical discharges from Malapterurus clectricus in and out of water.

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Figure 3. Ilorizontal projection of the crauial nerves of Electrophorus. Note the small size of the brain. Sone nerves are indicated by fhe corresponding numbers. L. A. nervus lateralis anterior;
. , L pcrvus lateralis postcrior; Oc-Sp, occipito-spinalis nerve.

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In our experimcnts, thc perception of objccts by weak clcctric fishes by way of the dischargc of thcir electric organs (transmitting and receiving) has bcen demonstrated. Other authors22 have shown that weak electric ßshes can be trained to distinguish betveen conducting and nonconducting objects placed in the water.

For this Ind of rcception, two possible modes of actioa at the level of spccific reand the "pulse-23
phase-modulation'23. According to the first hypothesis, sensory information should be conveyed by the frequency of the sensory impulses dependent on thc pulse of the electric organ discharge whereas, according to the second hypothesis, tho time relation (the phase) between the electric digcharge and the following scusory impulse would play an important role for the sensory coding. Frcm our exporimcnts with Sternarchus albifrons, a South American weak fresh water electric fish, we concluded that apparently reither of the above proposed mechanisms arc operating in this fish. The intensity of current flowing at the level of the receptor is coded by the number of impulses elicited by eaci elcctric organ discharge. St:rnarchus albifrons can discharge at rates higher than 1000 per second and thc sonsory impulses of the receptors can follow their dlischarge rate.

24 25 In some mornyrids\*and gymnotids, however, modulation of the rolation electricorgan pulse-sensory impulse may bc used for electrosensory coding. In mormyrids, also, the change in intensity of the electric field may be codod by charging tho latency between the electric organ pulse and the scnsory receptors' impulse. In mormyrids, it may attain values up to 9 mscc, whereans in gymnotids it has been found not to excced 1-2 mscc.

It has been previously mentioned that in Stcrnarchus albifrons the elc:croreceptors are distributed over {he entire body of the fish and that the ampullar,: onic receptors are more numerous than the tuberous phasic reccptors. We recorder ¿ien the autorhythmic clcctrical activity of the nonsynchronous tonic, anpullary c:zztrorcceptors of Sterarehus albifrons. The impulses were irregular aroud a rep:on rate of

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betwvccn 100 and 300, with an amplitude of around 2.5 mV. The impulsc duration was around 200 microscconds. There are other types of tonic reccptors which are synchronous. The phasic units are nonsynchronous.

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

As previously mientioned, some fresh water weak slectric fishes have the ability of perceiving objects, thcir movement and direction, and also to determine some characteristics of thcse objects (such as conductivity). For this underwater pattern recognition, they utilize both thcir electric transmitting organs and their electroreceivers. Obviously some other sensory percepton receivers of the lateralis line may be involved such as: free neuromasts and the zupula 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 species and also prey or predators. They use little: electrical encrgy for this and the distunce involved is ccnsiderable -- it nay attain, under certain circumstances and for one spccilc scnsitive species (Gymnarehidae), over a milo.

The strong clectric fishes (Elcctrophorus, Malapterurus, Torpcdo, and Astroscopus) can incapacitate and kill their prey with thcir ischarge of the powerful electric orgun used only for such purposcs. I thcy have a detccting and locating system, they use a different low power clcctrie transnitting than the main powerful electric orgu (i.c., Electrophorus). The mcaning of this is that there is no high power requirement for the pattern recognition system ef electric fishes. The clectric

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

The importunce of such an underwater pattorn recognition system cannot be overemphasized. The simulation and modeling of it can be achleved once the parameters of the differcnt sensory rcceivers have been estahishcd, physical analogs derived, and models devised.

By and large, the communication and coding systein of electric Gishes has been 27
de:onstrated tirough 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.

Lecause of our previous findings that the anaesthetic "MS 222 (Finquely" used by most researchers of electric fishes affects the frequency of the impulses emitted by the clectric organs in a nonuniforn way, we investigated a mumber of different anacsthetics and found one which does not affect the frequency of the impulses or their amplitude.

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II. INSTRUMENTATION

The recordings of the autorhytmic eicctr'· signals from electroreceptors nced special instrumentation. The signals are f low voltages: less than 1 mv, and they need a large bandwidth. The diameters .: some .f the electroreceptors are of the order of microns. There is the need for sulated microelectrode with a tip of one micron, with a reasonable low resistance, ., sibly less than one inegohm, responding from DC to several kHz with low ,wrtion and enough rugged to withstand some bumping by the fish. It has also tc e of a nonpclarizable 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 deve-oped a sensitive low roise 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 amplifi-. cation 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 óf the method used to produce a suitable microelectrode for recording electric signals from the electroreceptors of Sternar chus albifrons.

lubedescribed how to make coated tugsten microclcctrodes, Wohlbarsht, et al,9 10 31
described glass insulated platinum microclectrodos; Grcen and Grundfest, et al'
used stainless stecl clectrodes. The stcel and tungsten electrodes have a fairly high
resistance (20 to 100 megohms) and all, including the platinum clcctrode, are polarizable. 32
Donaldson describes a multitude of mieroclcctrodes sucl: as silver-silver chloride,
platinum-platinum chloride, and cthers. Silver-silver chlorides are very convenient

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elcctrodes but have a high resistance. In order to lower the resistance, it is advisable to cover the silver-silver chloride clectrode after the electrode is insulated with insulex lacquer, with platinum 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 witb Xylene (about 1/2"). Use a carbon rod for one electrode (spectroscopy carbon rods are suitable) and connet 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 p and down for about one minute (Figure 8). Decrtase 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 $\scriptstyle { \mathfrak { N a } } _ { 2 } { \mathsf { c o } } _ { 3 } ) ,$ (2) acetic acid 1% in $\mathfrak { F } _ { 2 } ^ { \mathrm { ~ O ~ } ( \mathrm { C H } _ { 3 } \mathbb { C } \mathrm { O } ) _ { 3 } - 0 , }$ (3) EthyI-alcolol 200 proof, (4) Xylene. Move electrode after sharpening process from No. 1 through No. 2, 3, and 4, agitating à few times the electrode 'n the liquid. Check under microscope with a microfilar for slarpness; 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 ${ \mathfrak { n } } _ { \underline { { \mathfrak { p } } } } 0 .$ Use a silver wire (No. 18 to 22 gauge) as an electrode (cathode) and connect it with a D. C. source (power supply) of between I to 2 voits. The positive end should be connccted to the microelectrode. Hold it for 30 seconds in the H Cl solution. Reverse twice the polarity for the samo amount of time (Figure 9). Wash the electrode in distilled water for two minutes. Insulate he elcctrode with insulex lacquer. For drying, set the microelectrodes with the tip up. Dry for 24 hours. The lip shoald be clean of Iacquer for 10 to 30 mieroas. Take a 50 ml beker and ll it with a 1% chloroplatinie acid. Use a No. 18 or 22 gauge wire as a cathode

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Éigure 9. Device for applying silver chloride on the tip and surface of silver or silver chloride platinized eleetrodes.

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onnected (to the negative) to a D.C. source of 15 volts. Fhe microelectrode should e connectcd 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 Ci 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 séconds will be sufficient. If bubbles come f 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 non-;olarizable and produces very little distortion from D.C. to fairly high freuency (over 1000 Hz) (Figures 10, 11).

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

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I. A SEARCH FOR AN ELECTRIC FISH ANAESTHETIC THAT WOULD NOT AFFECT THIEIR ELECTRIC SIGNAL

A commonly used anaesthetie for fishes and cold-blooded animais, urethan (ethyl carbamate $= \mathrm { C } _ { 3 } \mathrm { F } _ { 7 } \mathrm { N O } _ { 2 } )$ has boen found to have carcinogcnic properties by Wood 33 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 for urethan. MS-222 (tricaine methanesulfonate $\mathbf { \Sigma } = \mathbf { C } _ { 1 0 } \mathbf { F } _ { 1 5 } \mathbf { N O } _ { 5 } \{ \Sigma \}$ was discovered by M. Sandoz during his search for a reliable synthetic substituto for cocaine (benzoil-35
methylecgonine $= \mathsf { C } _ { \mathbf { 1 } \mathbf { 1 } } \mathtt { F } _ { 2 \mathbf { 1 } } \mathtt { N } \mathsf { O } _ { 4 } )$ 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 Marking®. Walker 37
and Schoettger measured its residues in various tissues of salmonides and its 38
efficacity has been investigated by Schoettger and Julin The chemical and anaes-39
thetic qualities of MS-222 are mentioned by Klontz, and details about the chemistry 40
can be found in the Merck Index

In the last years (1970-71), an agreement has been made between the Sandoz Pharmaceuticals, Division of Sandoz, Inc., Basel, Switzerland, and Hanover, N.J., USA, and the Ayerst Laboratorics, New York, N.Y., that thc latter shoult' produce and sell the MS-222 (tricaine methancsulfonate) in the United Statos under their own brand name of "FINQUEL".

In most of the sadies on elcctric fishcs, MS-222 has becn used as a gencral anacs-41 42 thetic by'the investigators. Szabo41, Hagiwara, Szabo and Enger42, Enger and Szabo43 , Nobuo Suga44, and many others used MS-222 to anacsthetize cleetrie fishes during surgieal procedures and subsequently to record tho clcctrical activity cither from the electroreceptors or from the nerves connecting t.em 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 elcçtrical signals as related to concentration and/or time. 45
Bullock mentioned that Hpopomus occidentalis, a South American fresh water weak clectric fish, witi a normal repetilion rate range of 25 to 90 per sccond, would lower its frequency to telow 16 per second only under anaesthesia. Under deep anaesthesia, the fish may stop abruptly its electrical activity. By reducirg 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 increese of the repetition rate with the recovery. By monitoring the effect of Finquel ("S-222) anesthetic on the electrical activity of Sternarchus albifrons, it has been. 'ed 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 cm tray made o lucite which could bc adjusted to the size of the fish has beon used to check the effects of anacsthetics on t.c clcctric organs of cleetric fish (Figure 12). The tray is provided with ittings for the rapid discharge of

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anaesthctics or vater and a constant acration of thc solution is possiblc if ncccssary. It also has at every ccntimeter distance embedded stainless steel electrodes connected on both sides of the tray with the exterior and to contacts. The elcctrodes corresponding to the position of the lead and tail of the Lish 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 roor and all the instrumcntation grounded to the screen room was located in the laboratoly outside the screened room. A lucite cover on the tray prevented the fish from jumping out, different holes served to pour in water or anaeshetics, to put in thermometers,and to let excess air out. A d-c (battery) operated high intensity lamp was used to illuminato from the top the fish and to furnish enough heat to hold the temperature constant during experiments. For the experiinents, the fishes' own acuarium-water was used at the beginning of the experiment and the same water was used for mixing in the anaesthetics. In this way, the tenperature of the water and anaesthetic solution was easy to keep to :he same level as the normal temperiture 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 experimnent.

Tentative measurements and observation of the action of the anaesthetic have been made first on goldfish and then on onc of the electric fisies before another specimen has becn selected for the experiment. For each expcrinent with one and the samc anacsthctic, five specimens of Sternarchus albifrons have bcen uscd. The specimcns of Sternarchus albifrons, a fresh watcr South American sternarchid wcak electric lish, have been in our laboratory for ovor one year and thcy were all healthy and varied in weight from 14 to 30 grains. Gynnnarchus niloticus, a fresh water African gymnarchić woak clectric fish,' has becn kept for over six months in our laboratory. There is another specimen just reccived. Tore aro aiso two Gnathoncmus petersii, fresh wate: Africaa mormyrid clectrie fish, being in the laboratory for over two months.

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The fishes have been weighed beforo each expcriment and put in the experimental tray in their own aquarium water. The electrical activity of the electric organ has been monitorel, recorded with the FM magnetic tape reccrder, 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 ono 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 magneti 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. T'he moment in which the fish was anaesthetized completely has been recorded. The fish respiration and electric activity (amplitude, wave form and repetitor ra'e) 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 recuvery 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 Iong 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 met'nanesulfonate, we rccorded tho electrical activity before, during, and after anaesthesia. The frequency of the elcctric organ vill drop immediatcly after adding the tricaine methancsulfonate to the water in a special tray,provided with stainless stecl clectrodes sct at a distance of ono cm from one another over the length of the lucite tray. The fish were restricted by a partition and a "U" shaped lucite device plced on top of the fish. Figure 14 shows the decrcase in frequency

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with time when the fish has bccn kcpt 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 an a Hewlctt-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 anaesthctic agents we tried, six were inadequate because the repetition rate of the electric sigmal was affected. Only the thiopental sodium Abbot (pentothal sodium) does not influence the electric activity of Sternarchus albifrons.

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 them repeatedly and after six months' time they are doing woll. 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 shów that the repetition rate of the electric signal has not been affectedl in over 40 minutes. The only change was produced by a slight decrease in water temperature (from 720 pulses to 700 pulses) of about 0.4 C. Sternarchus alhifrons is very sensitive to changes in water temperature. It will increase the repetition rate of the elcctric signal for an increase in water temperature and it will decrease the repctition rate for a decreaso in water tempcrature. For every degree Centigrade, it may change the repotition ratc by about 50 to 80, depending on the particular fish.

Tluopental sodi..m or Sodium 5-cthyl-5-(1-methylbutyl)-2-thiobarbitirate has the chemical formula $\mathsf { C } _ { 1 2 } \mathsf { \Pi } _ { 1 7 } ^ { \mathsf { H } } \mathsf { N a N } _ { 2 } \mathsf { O } _ { 2 } \mathsf { S }$ ,amolecular wcight of 26-1.3nd is a yellowish-

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whitc, hygroscopic power, solublc in water and alcohol and is a strongly alkaline 46 solution. The Abbot preparation is of nonhygroscopic crystuls -

Tubocurarinc chloride can be administered by intra-abdominal injection. 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 refrigeraton 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 ${ \bf C _ { 1 0 } } ^ { \mathrm { ~ \tiny ~ H ~ } } { \bf 1 5 } ^ { \mathrm { N O } _ { 5 } \tilde { s } }$ 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.

Fiuquel 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).

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Figure 28. Stcrnarchus albifrons No. 2 in MS-222 (Finqucl) Anaesthctic Solution 1:10000 in Water. Elcctric Signal Repetition Rate Afte 10 Minutes in thc Anacsthctic: ≈ 410.
Amplitudc 38 mv, swcep frcq. 1 mscc/em, water temp. 22.4°C, -8/31/72.

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IV. TECHNICAL DISCUSSION

Four specimens of Sternarchus albifrons have 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 Sternarchus albifrons.. Hagiwara, et $\mathrm { { a l } } ^ { 4 2 }$ , recommended 0.05 to 0.1 mg curare/fish and Enger and Szabo43 recommended 0.03 mg d-tubocurarine/g fresh fish weight. Both used (MS 222) tricaine methancșulfonate for anaesthiesia (1:150, 000). We found 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-abdominally. 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 ıl beaker with medicated aquarium water at a pH of 7. 0 and a teperature of $2 3 . 0 ^ { \circ } C$ . An aerator stone provided the necessary air.

A second specinen of Sternarchus (also 16 g) has been anaesthetized with MS 222, 1:25,000. One gram ofMS 222 has been dissolved in 1000 ml distilled $\mathtt { H } _ { 2 } \mathfrak { o }$ as stock solution. Then this was diluated to the proper amount in a 4000 ml beaker by adding aquariun water with merbromiue and acriflavine added as disinfectants. The MS 222 dilution of 1:150, 000 would not affect the fish in over oue hour. The dilution of the MS 222 was reduced to 1:25,000. After 20 mirutes 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 three minutes. For one hour it gave a good strong elcctric signal of the maip clcctric organ, after. this gradually it diminished in strength and in another hour the fish was dead.

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clearly the data given by the previous authors did not correspond for our specimens.

The third specimen of Sternarchus (18 g) was first anaesthetized in MS 222 1:12,500 for 20 minutes after this it was injected intra-abdomiaally with 0.1 ml o[ 0.05 mg/ml d-tubocurarine (Figure 31). The fish has been placed then in a special recording tray with circulating acrated water with merbromine, acrifavine and MS 222, 1:75,000. Recording from the electroreceptors has been attempted with a neutral electrode in the tray water and a silver-silver chloride microelectrode placed on a tonic, ampullary electroreceptor (Figure 32). The strong electric signal from the main transmitting electric organ masked the weak autorhythmic activity of the electroreceptors. This specimen has been returned to ts tank after 30 ninutes and rccovered almost instantly and is doing well since then.

Another fourth specinien of Sternarchus (19 g) receivcd the same treatment as the third specimen. This time instead of using the neutral electrcde 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. Alter 40 minutes, the fish was retirned to its tank and recovered almost instantly, liko the previous specimen, and is doing fine without any ill effects cince then (Figure 3H).

The microelectroce rccording experiments wcre porformed in a shiclded room from which all a-c instrunents, plugs and cords were eliminated and thc shiclded room then constituted a floating ground. llumination of the specimen was done by a microscope lamp fixed on the sterco-mieroscope and connceted to a 6 volt car batter'. All heaters were taken out of the water and all amplifiers wero connected to batteries. Other equipmient, such as the microscope support, the microscope, tho jack, the

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Figure 33. Set-up for recording autorhythmic activity of ampullary electrorcceptors of Stcrnarchus albifrons. The silversilver-chioride wire loop neutral electrode is visible.

Figurò 34. Anacsthetized,curarized Stcrnarchus albifrons in the recording {ray. Carbon eicctrodes record the activity of the transmitting organ as scen on the oscilloscope.

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amplifiers, etc., was comnected to one point ground and to. the Tektronix T22 modified differential amplifier ground. The microclectrode amplifier and support was changed from the integrated circuit model used before to an electrometcr tube type and a three transistor amplifier which is lcss sensitive to the change in impedance produced by slight movements of the ish than the previous one. The microelectrode, a silversiler-chlorido, 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 schomatic of the microelectrode 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 connected to another Tektronix T22 amplifier and displayed as a second trace on the scope. A Hewlett-Packard four clannel FM tape recorder was used to recorć 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 electric activity of & nonsynchronous phasic electroreceptor. After finishing the experiment, the fish was injected with neostigmine methylsulfate $\boldsymbol { 1 } { : } 1 0 ^ { 6 }$ to countcract the effect of the D-tubocurarine. The fishes recuperated in a few minutes and are doing woll.

Another experinient was perforned with two Gymnarchus niloticus, Cuv. Each of these fishes was placed in 25 gallon water tanks with Jucito trays raised to approximatoly 4 inches from the top. Gymnarchus niloticus is an air brcather and if lcft in a deep tank must expend excessive enorgy to swim to the surface for brcathíng. The tank is kept clcan by lucite plates provided with holcs to lct dlirt fall to the botlom where it can be vacuum-clcancd vory casily through spocial holes. The tunks of

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Gymnarchus niloticus are approximately thrce feet apart. .The covers and outside ponmagnctic stainless steel fraies were all grounded and so was the tark through carbon electrodes placed in the filters.

Two carbon electrodes in lucite tubes (with holes) werc placed at a distance of 15 cm from each other alongside the shes. A third electrode was placed either midway between the reccrding electrodes or on one of the ends of these electrodes. The first two electrodes for each fish were connected with the T22 Tektronix amplifiers and connected to two different Tektronix scopes. The third electodes were connected to a DPDT switch which could actuate also a battery to raise the lower trace of the scope when the two electrodes in the two tanks were connected and hence a communicason link was established between the two Gymnarchus to study the effect of social nteractfon 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 ten to twenty seconds. Figures 43 and 44 show the modified activity of the im-Ases froa Gymnarchus No. 3, and Figure 45 shows the modified activity of Gymnarchus o. 2. Wnen 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 betwveen o Gymnarchus niloticus.

e Gymnarchus niloticus baby was sectioned and fixed in buffered formaldehyde (10) about one month. Thcn it was decalcificd with Kristensen's decalcifying solutions 24 hours. After this, it was dehydrated using cthyl alcohol, toluene, tolucne with affin, and finally embedded in degasscd paraflin with 56°C mclting point.

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An A. O. Spencer microtome has been used to cut 10 micron slices. They were stained with Hematoxylin-Eosin and mounted with Permount on microslides. There are about 27 microslides with four to five serially cut transversal slices numbered from the head in the direction of the tail: 1-1, 1-2, 2-1, 2-2, ..., 7-3, 7-4. A few photomicrographs are shown as Figures 46, 47, 48, and 49. The interpretation of the histological preparation will be made at a later date. (Figure 50)

Malapterurus elcctricus, the clectric catfish (Figure 51) is diffcrent from the other :lectric fishes because it docs not obey Pacini's law. All tho othcr elcctric fishcs bey Pacini's law, according to which the innervated faccs of the clectroplates be-47 ome negative during the discharge, whatever the anatomical oricntition of the organ. his fact is due to the unique anatomy of Malapterurus. The clcctric organ of alapterurus forms a sort of loose jacket around the fish, instcad of being embekled

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Figure 46.Gymnnrchs niloticus, Cuv. (baby) Transversal cut through the last 1/1 in the direction of the tail.

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thin the body muscle as in all the othcr elcctric fishes. The clectric organ, depend-; on the fish's sizc, forms a sheath from 2 to 10 mm or cven thicker, in which the ctroplates are stacked in the longitudinal direction arranged in a somehow nonlerly fashion, contrary to the othcr elcctric fishes. The elecroplates are disks about 1 mm diamoter 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 myclinated ve fiber arising from a large ncrve cell situated in the gray matter of the spinal cord ween the levcls 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 could discharge impulscs 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 (approximtely 2 to ot) electric catfishes which the aquarium had did not discharge more than 100 volt es.

surprising that the Malapteruridac we checked could put about 0.5 w/hr of energy :ach gram of elcctric tissue of the olectric organ. It is our intention to investigate fact morc closcly. The electric discharge of three strong fresh water clectric s Malapterurus clcctricus woighing betwvcen 55-65 g was mcasured 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 mscc duration (Figures 52,53, 5-1).

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

The multimodulation-multicoding communication system of olcctric fishes served as a nodel in the developmcnt of a communication techníque to resist jamming.

he physical anclogs of a phasic and a tonic electrorcceptor have becn established for re Sternarchus albifrons, a South American fresh water weak electric fish of the high equency type (Figures 55, 50).The electroreccptors of other species have to be vestigated and the rclationships of tho various olectrorcceptors to tho undorwater ttern recognition systcm willbe established.

mentioncd tho cffect of moving objccts past the clectroreceptors in one or another :ection. Some of them would respond in onc way for, the fo:warà direct (head to tail) incrcasing the raie of the inpulses and in another way for the backvard direction

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current and voltage $\mathbf { r _ { g } }$ generator intcrnal resistance

external resistance $\mathfrak { a } _ { \mathfrak { a } }$ autornythnic generator

external canal resistance ${ \pmb { x } } _ { 1 } , : { \pmb { x } } _ { 1 2 }$ internal resistance

external canal capacitance $c _ { s }$ internal recuptor capacitance

receptor inner facc rcsistancc $\pmb { \tau _ { s } }$ skin resistance

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Figure 56 Phasic, tuberous electroreceptor. Physical analog.

current and voltage $\mathbf { r } _ { \mathbf { g } }$ generator internal resistance

external resistance $\mathbf { \sigma } _ { \mathbf { a } }$ autorhythmic generator

internal canal resistance $\mathbf { r _ { i _ { 1 } } } ; \mathbf { r _ { i _ { 2 } } }$ internal resistance

internal caual capacitance ${ \tt c } _ { \tt s }$ internal receptor capacitance

receptor cell capacitanco $\mathbf { r _ { s } }$ skin resistance

receptor inner face resistance $\sigma _ { \mathrm { e s } }$ external skin capacitunce

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(tail to head) by decreasing the rate of impulscs. Other electroreccptors will rcact to higher or lowor conductivity than the medium (aquarfum water) in a similar way. Obviously, the rcsponse of the electrorcceptors in othcr electric fish specics have to be asscssed in order to get a better overall picture of the underwator pattern recognition process. Figures 57 through 63 show two diffcrcnt elcctrorcceptors of electric fish. The results obtained from the electric receptors study wil lead to a simulation plan of an underwater pattern recognition system. A passive hybrid underwater pattern recognition system biock 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 senso:y 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 neurcmasts. 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, thrce sense organs are located between two pores. All :hree cupulae lie in a 'watery" 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 middlc unit is not in direct continuity with the external enironment (Ref. 23). It should be mentioncd that Gymnarchus as well as Notopterus :ave a completely closed canal system. Tho free ncuromasts found in the hoad of lernarchus 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 ymnarcbus Ref. 25), these free organs of Sternarchus are inncrvated b a bundle

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59. Ampullary tunic electroreceptor. (Schematic)

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

61 Tuberous phasic clectroreccptor. (Schematic)

Fig. 62 Slimulus and recording from a tubcrous phasic clcctroreccptor.

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c: tuberous electroreceptor type c. (Schematic)

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EL I to IV = ELECTRIC SENSORS ME I, ME = MECHANICAL SENSORS DISPL I, DISPL II = DISPLACEMENT SENSORS

FIGURE 64 PASSIVE HYBRID UNDERWATER PATTERN RECOGNITION SYSTEM

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ick and thin fibers. The specializcd lateral line organs (elcctric scnsory organs) be subdivided into to main groups: ampullary organs and tuberous organs. :e are many subdivisions of those organs and we havo previously reported about

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 theso organs that these "pores"should not be considered as I hole in the integument in every case, but rather a local differcntiation of the rmis overlying the sensory organs.

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V. RESULTS AND CONCLUSIONS

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

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

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

rdings and histological evidence show that Sternarchus albifrons has three electroreceptors: ampullary tonic nonsynchronous units, ampullary tonic hous units, and tubərous phasic nonsymchronous units. The physical analogs ind phasic electroreceptors arc shown in Figures 57 and 58. 3oth arc reprey a generator connected to rcsistances and capacitances in serics and in

The difference botwcen tonic and phasic electrorecceptors is that tho first e one resistance in scries with the generator whereas thephaslc elcctros have a capacitance. The tonic cloctroreceptors sccm to be predominant, ke five-to-one, compared to the phasic electroreceptors. The clectrorcseem to act, to a certain extent, indopendently of the main clcctric transmitting least two out of three different tpes of clec trorcceptors are asynchronous

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igure 66. Microelcctrode recording of e autorhythmic clectrical activity of ie ampullary, tonic elcctroreceptors of ternarchus albifrons. The spikes seen n the top of the rhythmic almost inusoidal waveform are the clectric iguals from the elcctroreceptors. orizontal: 1 graduation = 2 mscc. 'ertical: 1 gradnation = 500 mV. mplification xl00, effective 1 graduaon = 5 mV. Spikc app. 2 to 2.5 mV.

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ne type of electrorcccptor 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 l capable of responding to conductive and nonconductive objccts placed near ody. It may affect the total capability in determining certain movements or a certain extent, its sensitivity in pat tern recognition.Some of the syn-. nic units are connected to one and the same nerve trunk part of the acousticosstem but connected to specialized big nuclei in thc brain.

triking fact about fresh water wenk electric fish, besides their spontaneous an, 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 llis 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.

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

of the electric f'sh pattern rccognition system would make it possiblc to models of the phisical analogs of the sensors could be intograted in object location, detection and identification. The range and scnsitivity of tho 1 be assessed and improvements could be made.

lely used anaestl:ctic,- "MS 222"or "Finqucl" (tricainc mcthanesulfonate)- :cts the repetition rate of the clcctric impulscs of the clcetric organ. It :o do a series of experiments on different anaesthctics to establish whether

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:e is one which would not affect the frcqucncy of the impulses. It has becn found , thiopental sodium (sodium penthotal) will not affect the frequency of the impulses is a safe anaesthedc 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 detailcd way. The connections betwveen the electroeptors, the differert nerves, nerve-trunks and the brain have to be investigated. In . way, their interrclationship could be established. Microeiectrode 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 fbers.

m the experiments with Gymnarchus niloticus, we concluded -that one fish would ogrize 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 electroreceptors and from the nerve fibers of narchus albifrons and of the newly reccived Gymnarchus niloticus.

; possible to simulate an equivalent sensory system responding to different stimuli erwater. A system with a double fcedback 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 rclationship transmitter-receptor; and (2) another represented by a variable frequency transmitting system responding to disturbances he field between transmitter and reccptor with a change of the frequency of the smitting elcctric organ. To this we could add an independent dual autorhythmic eptor system: (a) responding with the increase or dccrcasc of the autorhythmic juency depending on movemcnt direction of the disturbance in lhe electric field; (b) responding with a change in the latency depending on the mignitude of the dlisanco, also distinguishing betwecn conductive and nonconductive objects.

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mnarchus niloticus, one of the most sensitive electric fishes, served as a model for simulation of a simplc pattern rccognition system (Figures 67 and 68).

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VI. REFERENCES

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he MERCK INDEX.

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Lartop: U. S. Patent 3109001 to Abbot Labs (1963).

\galides, E., Sensitivity and Behavioral Reaçtion of Sharks to Electric timuli, Final Report. ONR Contract Nonr 4773(00) No. 104-863 (1967).

lagiwara, S., Szabo, T., and Enger, P.S., Electroreceptor mechanism in 1 high frequency weaky electric fish, Sternarchus albifrons. J. Neurophysiol. 28:5:784-799 (1965).

Szabo, T., Nature, 194:4848:600-601 (1962).

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