INVESTIGATION OF ELECTRIC FISHES FINAL REPORT PHASE I by Prepared under: Contract 28 january 1974 SUMMARY The African fresh water *veakly electric fish Gnathoiiemus petersii has been investigated. The study has been difec-ted toward the intradermal sensory system with emphasis on the electroreceptors. Three types of electroreceptors have been identified. The autorhymic activity of2 these electrore- ceptors has been recorded. The variation of the electric signal of the electric organ have been recorded for three specimens of Gnathonemus petersii as a rest activity and maximum signal rate when a metallic object has been placed near the fish. The number and density of different kinds of electroreceptors in the dermis have been c9ounted and plotted against rate chanoe and their sensitivity to a metallic object. The preparation of the large tank experiments have been re- ported and the newly developed instrume,litation is mentioned. CONTENTS 1. INTRODUCTION .............................. 1 2. METHODS AND INSTRUMENTATION ................ 2 3. RESULTS ................................... 8 4. LARGE WATER TANK PREPARATION FOR EXPERIMENTS ............................... 26 FIGURES Figure Page 1 African fresh water wealcly electric fish Gnatlionemus petersii .................... 3 2 Electric fish Gnathonemus petersii in a luc2ite restraining tray provided with stainless steel electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Microelectrode amplifier . . . . . . . . . . . . . . . . . 5 4 Microelectrode amplifier and support in its shielding tube . . . . . . . . .2 . . . . . . . . . . . . . . . . 6 5 Microelectrode amplifier ready to be put in the shielding tube, face side . . . . . . . . . . . . . . . . . . 6- 6 Microelectrode amplifier ready to be put in the shielding tube, back side . . . . . . . . . . . . . . . . . 7 7 2 Microelectrode amplifier input and output wave- form and gaiii (see Table 2) . . . . . . . . . . . . . . . . 11 8 Microelectrode amplifier input and output wave- form and gain (see Table 2) . . . . .. . . . . . . . . . . . 11 9 Micro2electrode amplifier input and output wave- form and gain (see Table 2) . . . . . . . . . . . . . . . . 12 10 Microelectrode amplifier input and output wave- form and gain (see Table 2) . . . . . . . . . . . . . . . . 12 11 Microelectrode a1mplifier input and output wave- form and n, in (see Table 2) ................ 13 iv e- 13 input ',nd outl)ut wav Wlicx.oeler,trode r,,Ibie 2) . @see 12 forin Ind 6mul ,, ,,plifier inllut Id output,,Vave 14 2 laicroelectrol @see Table 2) 13 forin and ga"' lilier input and outl)ut wave- 14 1,Aicroelectrc,de OI-L"P le 2.) 14 and gain (see Tab d output wave - 15 ,ode arnplifier input a2" laicroelectr Table 2) - - - * 15 form and g@Lin (see ,t,,t wave 15 ,,,,,,plifier input and OA! 6icroelectrode (see Table 2) 16 forin and ga"' ut and output Nvave- 16 Mic,roelee,trode g@Lin (see Table 2) 'Output wave- 11 forrn and lifier input and 16 trode arnp Microelec 18 forin and @ain Gnathonernus is orgo@'n (electroreceptor) Of Tuberous 19 20 petersil atllonei-aus -y fields of G The electric sens2or 20 petersii - - - I I . . . r-y fields Of 20 rs senso the electrorecepto. . l'imits of li 21 nathonenius peters" tuberous G 2 Lst of raorrnyrorna-s -rnorrayrom@- 21 Different types orrayrorlast C. k-rfl 22 or gani b' ,view) (top and cut 2 the electric fish 22 lateral line nerves of 23 The Lljonernus petersii of the Gna: @ and density 0 etwee sel"sitiviti us petersii in the 24 Co 3 Itllone n" C tors of 24 ele epidern'lis v Figure Page 25 Autorliytmic activity of the electroreceptors of Gnatlionemus petersii: a. 500 Hz calibration signal., b. el(,,ctroreceptors near the chin, c. electroreceptors nea2r the eye . . . . . . . . . . . . . . 24 26 Electric activity from the nervus lateral anterior innervating receptor near the proboscis of a mech- anical displacement on the chin of Gnathonemus petersii when the proboscis has been moved upwards: a. time mar2ks = 50 Hz, b. electric activity in the nerve, c. movement of the chin proboscis . . . . . . 25 27 12 ft. diameter fiberglass tank provided with heat- ing, filtering, countercurrent aeration and double rails for electrode support . . . . . . . . . . . . . . . . 2 27 28 Heating tank provided with automatic control of temperature to 0. 0 IOC . . . . . . . . . . . . . . . . . . . 27 29 Differential amplifier hanging over the water tank . 28 30 Close look at the differential amplifier used in con- junction2 with the electrodes in the water tank to record electric activity of electric fishes . . . . . . . 28 31 Differential amplifier with remote control. Ampl. factor = x 4000, noise = 1 microvolt . . . . . . . . . . 29 32 Devices for restraining electric fishes in the water 0 tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 vi 1. INTRODUCTION In our letter report of 24 July 1973 we mentioned our interest in the moriiiyrid electric fishes. One of our reasons is that they have an electrical quantifiable behavioral variable. The rate and amplitude of their electric signal changes when they are electrically stimulated or. discontinuities ap- pear in their elect6romagnetic field. In our study of their electroreceptors we found some mechanoreceptors on the chin havinr a close morphology to the Lorenzini ampulla, a multisensory receptor. This fact confirms our findings with respect to the Lorenzini ampulla functions from our previous research. kND INSTRjMr,,NTATION Mr _IODS T, niorln-yrid Iresl" peltersii cxperiraents 2 oi Gnatholien-lus ,,, used ill Our Three ,CcillieTis ric I have bep- I-, one held se- iisll fr oil' .,Is and ear- Nveaillily ccctric 2 tor three Tnont ound ra. 9 ,d the ten" - wate were in Our laboratory 11 was ar The lisl-ies 1, ,,,lolls aq,,a-riO,, The water P paratel'Y nbedded in 2 ap c ont .2ec. 'OLets hall el pero,ture was it'h stainless steel P es during eneriynents lor as 1,,C-te tr-ay "v 2 J ('Fig. 2). I. 3sed to restrain the fish etric activit, lucite 'Wall %vere ol their ele the its of the repetition rate . ,,,,y developed'aad a, d- @essing the iim 2 lifiers prev3,0 red, the 0"set POte"t'o- -,ctrode Starlce Orclv raicroele croelectrode The ut resi .es ill2 the i:nP itive a-ad delicate rn' metirnes when chana Vienver-Y sells entioraeter was so Inirrons- ,et- e oliset Pot Of (. 5 rneter had to be res resetting Of th 2 ol the microelectr ode ode recordings -were made, the diarneter t Support or the Inicroelectr sible lob Owing to dire ,able tor the building, 01 r is a's o the ava 2 ira an impos de 3.MP""e nothing PL The Inicroelectro recently ecs. in elY until tae necessary 2 SP low output Tjllfortun'at inc, all actor) D. ?er- hlav v 1: fication pro, ,-,,Vl_iier I 2 . . ut re- sona,ble 3.1-i"P hanl@ inp nicroelectrode ,,,citance, a rea . tivity to c e in d have . h low ens' Ittle power all vlt low noise lactor a-id .. ins o bad to use pedalice N avo ver,Y It als s quor e w or lr ol iln2-pedallce to I IlleghonI. tion rate -lium 3 dB - sistance frOR 50 0 DC to 50, 000 repeti within Iroll-I t,on j, I r Cl 2rnean -y r- 'e _tctor Oul ,,ue,jr M', -niell a Ire with all an-1131ilic"l 2 p@Ic to DC or DC e(led ill building Su SUCCC conditions ol sn 11 2 lost, We un(ler ,dverse ,,,Ssitll recordn S 2 Fig. 1. African fresh water weakly electric fish Gnathonemus petersii. 7-- Fig. 2. Electric fish Gii,,ttlioiic@iiius petersii iii .1 lucite restr.,iiiiiiig tray provicied with si-.L-iiilcss steel 3 si@,nials from the clectrorcccl)tors of electric fislies, 'Ind wliicii does not need to reset the offset potentiometer. The .tmplifiers we used until now were the best which could be built, but they were far from the cal),,tbilities of the new microelectrodo amplifier, which incidentally, could be used in our simulation 6of the electric fish capabilities because of its low noise and very large bandwidth, combined with an insensitivity to change in input im- pedaiice. Figure 3 shows the schematic of the new amplifier, and Figs. 4, 5 and 6. are actual photos of the amplifier. 4 102.4 LODO oz 1< 10-2 K 2 RO u 2 R Tt!t TA6 I t4oTE Pla I U.IJDE)r@ f!f TAb --LECTI@ODE AMPLIFIF.'R eio t4. a.nd support 4. Microelectrode arnplifier in its sliielding tube. 4 0 icroelectrode -,,1,11)]Lifier re-,idy to be I)ut Fig. M tic si,iolding t'Llbc, ,,tcc side. Fig. 6. Microelectrode amplifier ready to be put in the shieldint tube, back side. 7 .. . ...... .... - - ------ - - -- ----- - ----- -- - - --- 3. RESULTS The specimens of the electric fish Gn,,itlioneniu.,; petersii were put in the lucite tray, using their own aquarium water and air was provided tlirouali • special glass tube. The temperature of the water has been recorded. After • few minutes acco2mmodation to their environment the electrodes correspond- in- to the head and tail of the fish, were connected to an amplifier, to the oscilloscope and to a counter. The rest activity has been read on the counter. Then a carbon steel rod (diam - 3 mm) has been immersed in the tray in the proximity of the fish. The repetition rate increased sicrnificantly and a read- ina of the counter has been made. Figure 2 2shows the fish in the tray. Table 1 shows the repetition rates of the signals. There is a ratio that could go to 1:23 (Fish No. 3) between the minimum and the maximum rate of the signal. Subsequent experiments could show how the repetition rate and amplitude of the sicrnals are related to the size, composition and proximity of the objects The microelectrode amplifier has been checked for its fre2quency and gain response using a Wavetek waVe,-,eiierator, atteiiuator and a Tektronix Oscilloscope type No. 555. The upper trace of the photos shows the input w,,tvcform and lower trace shows the output waveform of the amplifier. Both sine waves and square waves have been used. Table 2 shows the waveform, amplitude, gain and input resistance. Six photos we9re made for 50 ohms input resistance and six pliotos were made for 1 niegolim input resistance. TAB LE 1 Rest and Maximum Repetition Rate of the Electric Signal of Three Specimens of Gnathonemus petersii Signal Date Weight Water Fish of of Fish Rest Max. AmplituT2e- Temp. oc No. Recording in Grams Rep. Rate Rep. Rate mv in Instrumentation 1 8/12/73 15 15 136 500 20 Amplif.: 100x 9 Oscil. Tek. 555 3 8/11/73 22 7 161 500 20 Frequ. Counter for all Record- 4 8/12/73 15 15 135 500 20 ings '.rAA.BLE 2 Microclectrode Amplifier: Input and Output Waveforms and Results Gain/cm Photo Sine Sweep No. @i-i. cm In Out Input Res. 2 6 1K 2 msec 1 mv 1 v 50 G 7 10K .5 msee 1 mv 1 v 5OG 8 100K 1 mv 1 v 50 CZ Gain/cm Photo Square Sweep2 No. -F'L cm In Out Input Res. 9 1K 2 msee I mv I v 50 Cl 10 IOK .5 msec 1 mv 1 v 50 CZ 1 1 IOOK .05 msec 1 mv 1 v 250 C2 Gain/cm Photo Square Sweep No. -F'L c m In out Input Res. 12 IK 2 msec 1 mv 1 v 1 meghom 13 10K .5 msee 1 mv 1 v2 1 meghom 14 50K 1 msec 1 mV 1 V 1 meghom Gain/cm Photo Sine Sweep No. J)- cm in Out Input Res. 15 1K 2 msec I mv 1 v 9 1 megliom 16 10K .5 ms(--c 1 mv 1 v I meghom 17 50K msec 1 MV 1 v 1 mcgliom 10 r7 -ell Fig. 7. Microelectrode amplifier input and output -,vaveform and gain (see Table 2). Fig. 8. Microelectrode .iniplifier input aiid output waveforni ,tiid g,.iiii (see T,-ible 26). mom 0 RIA oulintnounn -0 DomoEmom Fig. 9. Microelectrodeamplif ier input and output waveform and gain (see Table 2). 3 I Ir@ @4 f.- -Z, L7 Fig. 10. Microclectrode .ti-nplifier imput and output wliveform ,tnd gain (see Ttble 2). 12 r; 6kJ -.,Mr F,. FL@". itoommig IL-1 Fig. 11. Microelectrodeamplifierinputandoutput waveform and gain (see Table 2). t:7 5 .47 Fig. 12. Microclectrode aniplifier input ,ind output waveforiii and g@.tin (see T,,tble 2). 13 @ Li @l;-i. .r3 -L7 Fig. 13. Microelectrode amplifier input and output Nvaveform and aain (see Table 2). 777 77: L Fig. 14. Mi3croelectrode aniplifier input and output w-,iveforiii and (see Table 2). 14 L rIl :-4i i,: I- ct Fig. 15. Microelectrode amplifier input and output waveform and gain (see Table 2). -4 0 Fig. 16. Microelectrode aniplifier input .ind output w,tvcforni tnd gtin (see Table 2). 15 rj Ficr. 17. Microelectrode amplifier input and output waveform and gain (see Table 2). L-7 L@l i Fig. 18. Microolectrode amplifier input and 2 output w,.,Lveform and gain. 16 For electric receptors we found two types of mormyromasts (A and B) and one type of tuberous organ. They are confined to well defined areas of the epidermis. The epidermis of these regions has a particular structure, which is developed in the Gymnotoides in a similar way. Its es- sential components are columns of very thin, flat hexagonal cells 60 4m in diameter, i2nvariable in all species and body sizes. The height of the columns depends on location., but increases with body length. The mormyromasts are not covered by the hexagonal cells, but by small polyhedric cells which are arranged in a circular pattern. The A-type mormyromasts possess an opening toward the surface and are evenly distributed with a relatively wide space between them. The B- t2ype mormyroi-nasts have no opening to the surface, and are more numerous than the A-type and are also evenly distributed. The tuberous organs lack an open connection to the surface and form distinct patterns (Fig. 19). They can be classified according to the number of their giant sensory cells (1 to 10). All mormyromasts and tuberous organs are innervated by lateral line nerves. Only the tip of the chin w2ith its Lorenzini ampullae is innervated by the Nerve trigeminus. Each mormyromast is enclosed by a loop of capillaries. The common lateral line system has developed only along the trunk and the tail. In the head only deep laying canals exist, but without sensory cells. The tuberous organs are characterized by an autorhytmic activity yielding a few mV. and with a high repetition frequency exceeding 1 kHz. The duration of the spikes are approximately 300 psec. The transmittinr electric org,,tn of Gn:,ttlionemus petersii is located in the tailstalk, occupying 2/3's of it and represents approximate i27o of the total length of the fish. 17 op- -7/ Fig. 19. Tuberous organ (electroreceptor) of Gnathonemus petersii. 18 The repetition rate of the impulses are influenced by light. In day- li-lit the rest repetition rate is between 7 and 10, in the night it increases 0 to 15-20. It will also increase considerably in the case of a stimulus affect- iiig the fish. The EMF of the fish Nvith no load and out of water is between2 7 and 17 volts depending on the particular specimen. The internal resistance is around a few kilo-ohms. The electroreceptors sensory fields of Gnathonemus petersii can be clearly visualized if we put the fish in a solution of 1001o buffered formaline. Figure 20 and 21 show the limits of these sensory fields. There are between 700 and 1000 tuberous organ 2electroreceptors, between 800 and 1000 type A mormyromasts electroreceptors and between 2100 and 2300 type B mormyromasts electroreceptors in the skin of an adult Gnathonemus petersii. The total number of electroreceptors varies between 3600 and 4300. These are distributed on the body as follonvs: between 42 to 460/o on the hc-ad on 41 to 440/o of the electrorece2ptor fields; between 30 and 327o on the dorsal sides on 27 to 30'0/o of the electroreceptor fields; and between 22 and 260/o on the ventral sides on 25 to 327o of the electroreceptor fields. The total area of the electroreceptor fields may occupy between 2000 and 5000 mm 2 area for fishes between 90 and 125 mm length. Figure 22 shows the different types of mormyromas2t electroreceptors of Gnathonemus petersii. With the exception of the sensory receptors of the chin which are mech- anical displacement receptors and are connected to the CNS through the Nervus trigeniinus, the mormyromast electroreceptors are subserved by the lateral line nerves. Figure 23 shows the main branches of the lateralis nerves system. All the mormyromasts types (tuberous, A and B) are connect8ed to nerves forniing bundles pertaining to the lateral line system and ending in the brain. 19 Fig. 20. The electric sensory fields of Gnatlio,.iemus petersii. Fig. 21. Limits of the electroreceptors sensory fields of Giiatlionemus petersii. 20 IV,, Fig. 22. Different types of mormyromasts: a. tuberous organ b. A-niormyromast c. B-mormyromast (top and cut view). 21 IT Oro h A( N.LAr.. iv rtA cr - 6 Fig. 23. Tile lateral line nerves of the electric fish Gnathonenius petersii. The tuberous orl@,,iii clectrorcccl)tors are itutorytliiiiic and the EMF may re-.icli ,i few millivolts. The repetition rate varies from 550 to 3900 with the most often encountered repetition rate between 0. 95 and 1. 95 kllz. Figure 24 shows -,t comp2irison between sensitivity and density of the electroreceptors in the epidermis of Giiathonei-nus petersii .iiid Fig. 25 sliows the autorliytmic activity of the electroreceptors near the chin and near the eye. FiLo,iire 26 shows the autorliytmic activity of the mechanical displace- ment sensory or-ans as an effect of bending the proboscis of the c8hin. Experiments in this direction would be continued to record wave- form and chan-es in repetition rate as a result of different stimuli. 23 Fig. 24. Comparison between sensitivity and density of the electroreceptors of Gnathonemus petersii in the epidermis. a vvvvw@ b 45 c Fig. 25.7 Autorhytmic activity of the electroreceptors of Gnatlionemus petersii: a. 500 Hz calibration signal b. electroreceptors near the chin c. electroreceptors near the eye 24 a..................................................................................................................... b c ........... ..................................... .............. -------------------------------------- .......... b C 2 s lateral anterior innervating Fig. 26. Electric activity from the nervu receptor near the proboscis of a mechanical displacement on the chin of Gnathonemus petersii when the proboscis has been moved upwards. a. time marks = 50 Hz b. electric activity in t5he nerve c. movement of the chin proboscis 4. LARGE WATER TANK PREPARATION FOR EXPERIMENTS The 12 ft. diameter, 4 ft high water tank has been prepared for the other experiments that will follow for the Phase H investigation (Fig. 26). Heating the water is done with 2 x 1000 watts heaters controlled by an IIYSI" temperature controller to ± . IOC and is normall2y held at 250 C. The heaters are in a separate 30 gallon tank and are connected to a relay switching them on and off and controlled by the temperature controller. Two 9 gallon per minute pumps are pumping in and out the water from the 30 gallon tank from and into the large tank (Fig. 27). Rails with nylon strings are provided for the silver -silver chloride- platinized-silver-chlorized electrodes 2which can be moved from one end to the other end of the tank (Fig. 28). The electrodes are connected to a re- mote controlled differential amplifier (ampl. fact. x 4200) suspended over thE@ tank and from the amplifier to the differential oscilloscope Tektronix type 555 (Figs. 29, 30 and 31). An electric fish can be suspended in one of the restraining devices shown 2in Fig. 32. The fish restraining devices are provided with stainless steelend electrodes which are connected to ,in audio-amplifier (ampl. fact. 300) and to the oscilloscope and displayed on a second beam. Our preliminary experiments show good promise for recording the ch,,inges in the field of electric fishes produced by them as a result of stimuli they are presented with. 4 26 ..It$ It Firr. 27. 12 ft. diameter fiberglass talilr- provided with heating, filtering, countercurrent aeration and double rails for electrode support. Aug $4 Fi(,-. 285. He.,tting tai-Lk provided with .ititoiiiatic control of temperature to O. O loc. 27 Aga Fig. 29. Differential amplifier hanging over the water tank. Fig. 30. Close look at the differential anii)].ifier ilsed in conjunction with the electrodes in the water tank to record electric activity of electric fislies. 6 28 Fig. 31. Differential amplifier with remote control. Ampl. factor x 4000, noise 1 microvolt. Fig. 32. Devices for restraining electric fishes in the water tank. 29