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Picrotoxin prevents habituation of the gill withdrawal reflex in Aplysial KENLUKOWIAK~ Depul.rmenr o f Physiology, McGill University, Montreal, P.Q., Canada
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/14/14 For personal use only.
Received August 16, 1978 LUKOWIAK, K. 1978. Picrotoxin prevents habituation of the gill withdrawal reflex in Aplysin. Can.J . Physiol. Pharmacol. 56, 1079-1082. The gill withdrawal reflex evoked by tactile stimulation of the siphon in Aplysin habituates with repeated presentation of the stimulus. This adaptive behaviour is mediated by the integrated activity of the central (CNS) and peripheral (PNS) nervous systems. The PNS mediates the basic reflex and its habituation while the CNS exerts both suppressive and facilitatory control over the PNS. This results in greater adaptability of the reflex behaviours. In young Aplysitz the CNS control is absent and this is due to the incomplete development of pathways in the CNS. In an attempt to identify the pathway an attempt was made t o manipulate the CNS's suppressive influence by agents which antagonize putative neurotransmitters. The application of picrotoxin-containing seawater over the CNS removed the CNS's suppressive influence but not its facilitatory influence. Thus the reflex amplitude was increased, the reflex latency decreased, and repeated stimulation did not result in habituation. This eEect of picrotoxin was completely reversible. It is thus proposed that y-aminobutyric acid, a putative neurotransmitter, plays an important role in the mediation of the CNS's suppressive influence.
Introduction Habituation of the gill withdrawal reflex in A plysia has been extensively studied in an attempt to gain an understanding of the neuronal mechanisms which underlie adaptive behaviour (Jacklet and Lukawiak 1975; KandeI 1976). The withdrawal reflex and its subsequent habituation evoked by tactile stimulation of the siphon, in the manner described below, are mediated by the integrated activity of the CNS and PNS (Lukowiak and Peretz 1977). The PNS between the siphon and gill mediates the reflex and its behaviour while the CNS exerts control over the PNS. As a consequence of this CNS control, the reflex behaviours are much more adaptable to changing stimulus conditions (Lukowiak 1977). The CNS exerts both suppressive and facilitatory control over the PNS-mediated behaviours. The suppressive control is mediated via the branchial nerve while the facilitator~influence is mediated via the ctenidial nerve. If the branchial nerve is severed, leaving everything else intact, the reflex latency is significantly decreased, the reflex amplitude becomes larger, and repeated tactile stimulation of the siphon no longer results in habituation (Lukowiak 1977).However, in young Aplysia the CNS control is absent (Lukowiak 19780). Removal of the branchial nerve input to the gill does ABBREVIATIONS: CNS. central nervous system; PNS, peripheral nervous system; GABA. y-aminobutyric acid. 'This research was supported by the Medical Research Council of Canada. 'Present address: Division of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alta., Canada T2N 1N4.
not affect reflex latency, amplitude, or habituation of the reflex. This difference between young and older Aplysia was found to be due to the incomplete development of neural pathways in the abdomir~al ganglion (CNS) and not the incomplete development of the PNS (Lukowiak 1978a). Attempts are being made to identify the neural pathways which mediate the CNS control and which are not yet developed or functional in the young. One strategy employed was to attempt to manipulate the CNS's suppressive influence by agents which antagonize the action of several putative neurotransmitters. This short communication describes the effect of the perfusion of picrotoxin over the abdominal on the latency amplitude and ability of the reflex to habituate.
Methods Fifteen Aplysin cnlifornica (Pacific Biomarine Laboratories, Venice, C A ) weighing between 150 and 250 g were used. The preparation used was similar to that previously described (Lukowiak and Peretz 1977; Lukowiak 1977) and consisted of the siphon, mantle, gill, and abdominal ganglion. The abdominal ganglion innervates the gill via the branchial, ctenidial, and a small branch of the siphon nerve. All other nerves and connectives were severed. The abdominal ganglion was pinned out on a piece of sylgard in artificial seawater (Instant Ocean) maintained at 15-16°C and p H 7.9. A small chamber, which was sealed with petroleum jelly, was fitted around the ganglion. A check was made that the chamber did not leak, thus the solution bathing the ganglion could be altered without affecting tile seawater bathing the rest of the preparation. The capacity of the perfusion chamber was 2-3 ml. Microelectrodes filled with 3 M KC1 and having a resistance of 15-30 MQ were used. The ganglion was transilluminated
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CAN. J. PHYSIBL. PHAWMACOL. VOL. 56. 1948
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to aid in the identification and impalement of neurons. Gill withdrawal anovenaents we1 e rneasuk ed using a G Iass tension tlansducer connected to a single gill pinnetIe by fine surgical thread. The ol~tputof the transducer was recorded on ;L ~ t o ~ n goscilloscope e and a poIyg~aphfroral which the mear~trernentswere made ( 7 nsm - - 200 mg) . Tactile sti~nuliwere delivered to the siphon by a Tapper' which is a piastic-coated wire, I rnm ian diarneler, which wns ccaranected tea a \olenoid (for ;i lalore complete descripticm of the Tappcr :md the recording techniques see Peret7 and Lukowiak (1975) and Lukowiak and P e r e t ~(1977) ) . The stimulus intensity chcasen for this series of experiments w a \ 1000 rng, the same inten\ity that ha\ been used previcdukly (Lethowiak 1977). An habituation series consisted of at least 1 0 pl esentalions of the tactile \tirnulus to the seghon with an interstirnult~sinterval of 30 s. Fach series was folIo\eed by a rest period o f 3 h wiaich allows foa- coanplete recoeTery ( P e r e t ~ and Howieson 1973; Lukowiak 1977). Each preparation served as its own contl-01. It w a s first testcd with seawater in the perfusion chamber and then 3 h later the seawater in the pel-fusion chamber was replaceel by n solution of picrotoxin ( 10 "-10 " M ) . Twenty minutes later the preparation after the second was again stimulated. In scme -prepal-ations, . habituation series the picrotoxin so1~atiol.rwas removed and replaced bjr seabater and 3 lh later the reflex was agaia-n habituated. The reflex latency and aanplitalde were obtained from the first trial of each hxhitaiation seriec. Thus, thc eflect of picsotoxin can be easily assessecl.
Results The effects of the perfusion of picrotoxi~lover the abdsnninal garmglicrn on the latency and arnplitudc of the gill witladrawal reflex were first examined (Fig. I ). In the 15 preparations tested the mean latency was 289 rns with seawater bathing the ganglion while with picrotoxin bathing it the latency was significantly reduced ( r test, p < 0.07 1 to a mean of 180 ms. Tlae perfaision of picrotoxin also significantly affected the arrnplitude of the reflex ( p < 0.05) ; the aralplitude irncreaseci by a mean of 127% over the control amplitude. Thus, the perfusion of picrotoxin s ~ ~ ethe r abdominal ganglion brought about sir~ailarefTects on the reflex latency and amplitude as did cutting of the branchial nerve (Lukowiak 1977). Tlae perfusion of picrotoxin over the ganglion had no systematic change on the activity evoked in gill motor neurons L7 or EBG,, nor did it have any observable effect OIP the periodicity of the spontaneously occearring periodic gill respiratory ~novennents. Picrotoxirm was also found to affect habituation crf the gill withdrawal reflex evoked by repeated tactile stimulation of the siphon. These data are shown in Fig. '2. As can be seen, the gill witladrawal reflex habituated raormally when the ganglion was bathed in seawater. However, wit11 the addition of picro-
F1cr.1. 'The effect of perfusion of picrotoxin a9ver the abdominal ganglion on the latencj and amp8itude of the gill ~4thcbtaualreflex es~okecbby tractile stimulation of the siphon. (A1 The gill withdtawal reflex evohed k y siplson \timuIation ( 1000 mg) with nc~rm;~I seawater bathing the abdominal ganglion. The tactile stimulus also evohed activity in gill motor neuron L,,. ( H ) One hour later and 20 min :ifher the perfusion of seawater containing 10 " .M picrotoxin over the abdominal ganglion. As can be reac%ilyseen, the latency of the reflex was significantly decreased. The black lines drawn in indicate the ti~laebetween the onset of the stirnralr~sand the onset of the gill c o n t i a c t i o ~ ~ As. can also be seen, there was littie or no reduction in the latency of the input to I.,,. 'The arnpiitude of the reflex in B was larger than the amplit~adeof the contraction in 1% (31 mn-n vs. 35 mna. as measured on the polygraph). Scales: L,,, 20 milliunits per cfivision; gill te~ssion,0.%/2milliunits; 100 ms.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/14/14 For personal use only.
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FIG.2. The effect of picrotoxin on gill withdrawal reflex habituation. (-4) Pooled normalized (lOD';( as initial response) data (tr = 15) showing the effect of repeated (1,'30 s) tactile stimulation of the siphon (1000 mg) on the amplitude of the gill withdrawal reflex. -4s can be seen, when the abdominal ganglion was perfused with normal seawater )-.( the reflex habituated while 3 h later and after 20 min in picrotoxin-containi~~g seawater, the reflex no longer habit~~ated ( :-n). (H) The data are as in -4, only they have not been normalired In order t o show that picrotoxin also affected the art~plitudeof the gill withdrawal reflex in addit~onto affecting the reflex latency and habituation. In both -4 and I3, the the standard error of the mean has been plotted. mean
+
Thus, when the abdominal ganglion was perfused with picrotoxin-containing seawater the reflex latency was significantly reduced, the reflex amplitude was significantly increased, and the reflex no longer habituated with repeated siphon stimulation. Picrotoxin appeared to act specifically on those neural pathways involved in the control of the defensive gill withdrawal reflex. The neural pathways which mediate the spontaneous rhythmic gill respiratory movements were not affected by picrotoxin as evidenced by no observable changes in either the amplitude or the frequency of these contractions. Thus, the effects brought about by picrotoxin were not only reversible but were specific to the neural pathways which exert suppressive control over gill reflex behaviours. Because the picrotoxill effects are both specific and reversible they may help in the identification and characterization of the neural pathways in the CNS which exert control over the PNS. The data obtained in this present study are similar to the data obtained previously when the branchial Discussion nerve influence to the gill was removed (Lukowiak The data presented here show that the CNS's sup- 1977). In that study, following ren~oval of the pressive influence on the PNS can be manipulated branchial nerve influence, the reflex latency deby picrotoxin at concentrations of 10 6-10-W. creased, the reflex amplitude increased, and the re-
toxin to the bathing solution surrounding the ganglion the reflex no longer habituated. Both the normalized and unnormalized group data have been plotted to show better the effect of picrotoxin on the ability of the reflex to habituate. There is no doubt that the addition of picrotoxin prevents habituation of the reflex from occurring. This effect of picrotoxin was completely reversible because in a number of preparations 3 h later the reflex once again habituated. Although not shown here, the presentation of a novel stimulus was much more effective when picrotoxin-containing seawater was bathed over the ganglion. The sensitizing effect of the novel stimulus resulted in reflex amplitudes that were facilitated by up to 250% of the initial response and this facilitated state persisted for 3-5 min. This phenomenon will be examined and reported in greater detail at a later time. Thus, picrotoxin not only had the effect of potentiating the reflex a~nplitudebut also preventing habituation from occurring.
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CAN. J. PHYSIOL. PHARMACOL. VOL. 56. 1978
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/14/14 For personal use only.
flex no longer habituated with repeated stimulation. These changcs in gill reflex behaviour were not accompanied by any change in synaptic input or change in synaptic eficacy to the gill motor neurons in the abdominal ganglion (Lukowiak 1977). Whether this will also be the case with the picrotoxin experiments remains to be determined. Picrotoxin may bring about its effects on gill withdrawal reflex behaviours by antagonizing GABA. Recently, Tremblay and Plourde ( 1977) have shown in Aplysia central neurons that GABA's actions are antagonized by picrotoxin at concentrations similar to those used here. Further support for this notion are preliminary data which show that in young Aplysia, where the suppressive C N S control is not yet functional (Lukowiak 1978a), perfusion of GABA over tlae abdominal ganglion results in a significantly faster rate of habituation. Thus, GABA appears to allow the suppressive influence to become functional (Lukowiak: in preparation). GABA may thus be of prime importance in the neural pathways which exert suppressive control of gill reflex behaviours. Picrotoxin has previously been used to attempt to distinguish between possible neural mechanisms of habituation. Habituation could be due to active suppression or it could be due to a decrease in synaptic efficacy intrinsic to the reflex's excitatory pathways. If habituation were due to active suppression, then picrotoxin which blocks known inhibitory processes should prevent or at least retard the rate of habituation. Using this argument, Spencer ct al. (1966) ruled out a buildup of suppression to explain habituation of the flexor reflex in the spinal cat. Krasne and Roberts (1967) similarly found that picrotoxin did not alter habituation of thc escape reff ex in crayfish. They concluded that habituation was due to a depression intrinsic to the excitatory pathways of the reflex. However, Glantz (1974) found that picrotoxin couId reverse a previously habituated response and delay the onset of habituation. He suggested that an inhibitory mechanism may play an important role in the mediation of habituation. The data presented here Iend further support to the hypothesis that inhibition plays an important role in the mediation of gill withdrawal reflex habituation in Aplysia. This is not to say that the syiaaptic decrement which occurs between the central sensory neurons and the central gill rnotor neurons in the abdominal ganglion which accompanies habituation of the gill withdrawal reflex is brought about by an increase in pre- or post-synaptic inhibition. On the contrary, the evidence to date (Castellucci and Kandel 1974) suggests that this decrement is the
result of an intrinsic property of those synapses. However, it is important to recall that this synaptic decrement continues to occur even though the reflex itself doesn't undergo habituation (Lukowiak 1977, 1978h). Habituation of this reflex is not due solely to changes which occur togethcr within the integrated CNS-PNS (Lukowiak and Peretz 1977; Lukowiak 1977). It thus appears that an increase in inhibition plays an important role in the mediation of habituation by the entire integrated system. Picrotoxin actions affect the output of the increased suppressive CNS influence brought about by repetitive stimulation of the siphon. The neural pathways which initiate this suppression are not known, nor is it known where or how in the integrated system this suppression is mediated. These questions remain to be answered. C A ~ T E L L UV., C ~and ~ , KANDEL, E. K. 1974. A quanta1 analysis of the synaptic depression underlying habituation of the gill withdrawal reflex in Aplysia. Proc. Natl. Acad. Sci. U.S.A. 71, 5004-5008. GLANTZ,R. 1974. The visually evoked defensive ref ex of the crayfish: Habituation, facilitation, and the influence of picrotoxin. J. Neurobiol. 5, 263-278. JACKLET, J. W., and LUKOW~AK, K. 1975. Neural processes in habituation and seilsitization in model systems. li'rog. Neurobiol. 4, 1-56. KANDEL, E. R. 1976. Cellular basis of behavior. An introduction to behavioral neurobiology. W. H. Freeman and Co., San Francisco. KKASNE,F. B., and ROBERTS, A. 1967. Habituation of the crayfish escape response during release from inhibition induced by picrotoxin. Nature (London), 215, 769-770. LUKOWIAK, K . 1977. CNS control of the PNS-mediated gill withdrawal reflex and its habituation. Can. J. Physiol. Pharmacol. 55, 1252-1 262. --- 1 9 7 8 ~ .Development of CNS control of the siphon evoked gill withdrawal reflex in Aplysicr. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 208. -- 1978h. Induced LD activity prevents habituation of the gill withdrawal reflex evoked by siphon stimulation in rlplysia. Soc. Neurosci., in press. L U K ~ W I AK., K , and PERETZ, B. 1977. The interactions between the central and peripheral nervous systems in the mediation of gill withdrawal reflex behaviour in Aplysia. J. Comp. Physiol. 117, 219-244. PEKETZ,R., and HOWIESON, D. 1973. Central influence on peripherally mediated habituation of an Aplysicr gill withdrawal response. J. Comp. Physiol. 84, 1-18. PPKETZ,B., and LUKOWIAK, K. 1975. Age-dependent CNS control of the habituating gill withdrawal reflex and of correlated activity in identified neurons in Apl~lsia.J. Comp. Physiol. 103, 1-17. SPENCER, W. A., THO~.IPSON, K. F., and NEILSON,I). 1966. Decrement of ventral root electrotonus and intracellularly recorded post-synaptic potentials produced by iterated cutaneous afferent volleys. J. Neurophy siol. 29, 253-274. TREMBLAY, J. P., and PLOURDE, G. 1977. Presynaptic modulatory effects of GABA on depression, facilitation, and posttetonic potentiation of a cholinergic synapse in Apljqsicr culijornicu. Can. J. Physiol. Pharmacol. 55, 1286-1 301.
Picrotoxin prevents habituation of the gill withdrawal reflex in Aplysial KENLUKOWIAK~ Depul.rmenr o f Physiology, McGill University, Montreal, P.Q., Canada
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/14/14 For personal use only.
Received August 16, 1978 LUKOWIAK, K. 1978. Picrotoxin prevents habituation of the gill withdrawal reflex in Aplysin. Can.J . Physiol. Pharmacol. 56, 1079-1082. The gill withdrawal reflex evoked by tactile stimulation of the siphon in Aplysin habituates with repeated presentation of the stimulus. This adaptive behaviour is mediated by the integrated activity of the central (CNS) and peripheral (PNS) nervous systems. The PNS mediates the basic reflex and its habituation while the CNS exerts both suppressive and facilitatory control over the PNS. This results in greater adaptability of the reflex behaviours. In young Aplysitz the CNS control is absent and this is due to the incomplete development of pathways in the CNS. In an attempt to identify the pathway an attempt was made t o manipulate the CNS's suppressive influence by agents which antagonize putative neurotransmitters. The application of picrotoxin-containing seawater over the CNS removed the CNS's suppressive influence but not its facilitatory influence. Thus the reflex amplitude was increased, the reflex latency decreased, and repeated stimulation did not result in habituation. This eEect of picrotoxin was completely reversible. It is thus proposed that y-aminobutyric acid, a putative neurotransmitter, plays an important role in the mediation of the CNS's suppressive influence.
Introduction Habituation of the gill withdrawal reflex in A plysia has been extensively studied in an attempt to gain an understanding of the neuronal mechanisms which underlie adaptive behaviour (Jacklet and Lukawiak 1975; KandeI 1976). The withdrawal reflex and its subsequent habituation evoked by tactile stimulation of the siphon, in the manner described below, are mediated by the integrated activity of the CNS and PNS (Lukowiak and Peretz 1977). The PNS between the siphon and gill mediates the reflex and its behaviour while the CNS exerts control over the PNS. As a consequence of this CNS control, the reflex behaviours are much more adaptable to changing stimulus conditions (Lukowiak 1977). The CNS exerts both suppressive and facilitatory control over the PNS-mediated behaviours. The suppressive control is mediated via the branchial nerve while the facilitator~influence is mediated via the ctenidial nerve. If the branchial nerve is severed, leaving everything else intact, the reflex latency is significantly decreased, the reflex amplitude becomes larger, and repeated tactile stimulation of the siphon no longer results in habituation (Lukowiak 1977).However, in young Aplysia the CNS control is absent (Lukowiak 19780). Removal of the branchial nerve input to the gill does ABBREVIATIONS: CNS. central nervous system; PNS, peripheral nervous system; GABA. y-aminobutyric acid. 'This research was supported by the Medical Research Council of Canada. 'Present address: Division of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alta., Canada T2N 1N4.
not affect reflex latency, amplitude, or habituation of the reflex. This difference between young and older Aplysia was found to be due to the incomplete development of neural pathways in the abdomir~al ganglion (CNS) and not the incomplete development of the PNS (Lukowiak 1978a). Attempts are being made to identify the neural pathways which mediate the CNS control and which are not yet developed or functional in the young. One strategy employed was to attempt to manipulate the CNS's suppressive influence by agents which antagonize the action of several putative neurotransmitters. This short communication describes the effect of the perfusion of picrotoxin over the abdominal on the latency amplitude and ability of the reflex to habituate.
Methods Fifteen Aplysin cnlifornica (Pacific Biomarine Laboratories, Venice, C A ) weighing between 150 and 250 g were used. The preparation used was similar to that previously described (Lukowiak and Peretz 1977; Lukowiak 1977) and consisted of the siphon, mantle, gill, and abdominal ganglion. The abdominal ganglion innervates the gill via the branchial, ctenidial, and a small branch of the siphon nerve. All other nerves and connectives were severed. The abdominal ganglion was pinned out on a piece of sylgard in artificial seawater (Instant Ocean) maintained at 15-16°C and p H 7.9. A small chamber, which was sealed with petroleum jelly, was fitted around the ganglion. A check was made that the chamber did not leak, thus the solution bathing the ganglion could be altered without affecting tile seawater bathing the rest of the preparation. The capacity of the perfusion chamber was 2-3 ml. Microelectrodes filled with 3 M KC1 and having a resistance of 15-30 MQ were used. The ganglion was transilluminated
1080
CAN. J. PHYSIBL. PHAWMACOL. VOL. 56. 1948
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/14/14 For personal use only.
to aid in the identification and impalement of neurons. Gill withdrawal anovenaents we1 e rneasuk ed using a G Iass tension tlansducer connected to a single gill pinnetIe by fine surgical thread. The ol~tputof the transducer was recorded on ;L ~ t o ~ n goscilloscope e and a poIyg~aphfroral which the mear~trernentswere made ( 7 nsm - - 200 mg) . Tactile sti~nuliwere delivered to the siphon by a Tapper' which is a piastic-coated wire, I rnm ian diarneler, which wns ccaranected tea a \olenoid (for ;i lalore complete descripticm of the Tappcr :md the recording techniques see Peret7 and Lukowiak (1975) and Lukowiak and P e r e t ~(1977) ) . The stimulus intensity chcasen for this series of experiments w a \ 1000 rng, the same inten\ity that ha\ been used previcdukly (Lethowiak 1977). An habituation series consisted of at least 1 0 pl esentalions of the tactile \tirnulus to the seghon with an interstirnult~sinterval of 30 s. Fach series was folIo\eed by a rest period o f 3 h wiaich allows foa- coanplete recoeTery ( P e r e t ~ and Howieson 1973; Lukowiak 1977). Each preparation served as its own contl-01. It w a s first testcd with seawater in the perfusion chamber and then 3 h later the seawater in the pel-fusion chamber was replaceel by n solution of picrotoxin ( 10 "-10 " M ) . Twenty minutes later the preparation after the second was again stimulated. In scme -prepal-ations, . habituation series the picrotoxin so1~atiol.rwas removed and replaced bjr seabater and 3 lh later the reflex was agaia-n habituated. The reflex latency and aanplitalde were obtained from the first trial of each hxhitaiation seriec. Thus, thc eflect of picsotoxin can be easily assessecl.
Results The effects of the perfusion of picrotoxi~lover the abdsnninal garmglicrn on the latency and arnplitudc of the gill witladrawal reflex were first examined (Fig. I ). In the 15 preparations tested the mean latency was 289 rns with seawater bathing the ganglion while with picrotoxin bathing it the latency was significantly reduced ( r test, p < 0.07 1 to a mean of 180 ms. Tlae perfaision of picrotoxin also significantly affected the arrnplitude of the reflex ( p < 0.05) ; the aralplitude irncreaseci by a mean of 127% over the control amplitude. Thus, the perfusion of picrotoxin s ~ ~ ethe r abdominal ganglion brought about sir~ailarefTects on the reflex latency and amplitude as did cutting of the branchial nerve (Lukowiak 1977). Tlae perfusion of picrotoxin over the ganglion had no systematic change on the activity evoked in gill motor neurons L7 or EBG,, nor did it have any observable effect OIP the periodicity of the spontaneously occearring periodic gill respiratory ~novennents. Picrotoxirm was also found to affect habituation crf the gill withdrawal reflex evoked by repeated tactile stimulation of the siphon. These data are shown in Fig. '2. As can be seen, the gill witladrawal reflex habituated raormally when the ganglion was bathed in seawater. However, wit11 the addition of picro-
F1cr.1. 'The effect of perfusion of picrotoxin a9ver the abdominal ganglion on the latencj and amp8itude of the gill ~4thcbtaualreflex es~okecbby tractile stimulation of the siphon. (A1 The gill withdtawal reflex evohed k y siplson \timuIation ( 1000 mg) with nc~rm;~I seawater bathing the abdominal ganglion. The tactile stimulus also evohed activity in gill motor neuron L,,. ( H ) One hour later and 20 min :ifher the perfusion of seawater containing 10 " .M picrotoxin over the abdominal ganglion. As can be reac%ilyseen, the latency of the reflex was significantly decreased. The black lines drawn in indicate the ti~laebetween the onset of the stirnralr~sand the onset of the gill c o n t i a c t i o ~ ~ As. can also be seen, there was littie or no reduction in the latency of the input to I.,,. 'The arnpiitude of the reflex in B was larger than the amplit~adeof the contraction in 1% (31 mn-n vs. 35 mna. as measured on the polygraph). Scales: L,,, 20 milliunits per cfivision; gill te~ssion,0.%/2milliunits; 100 ms.
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FIG.2. The effect of picrotoxin on gill withdrawal reflex habituation. (-4) Pooled normalized (lOD';( as initial response) data (tr = 15) showing the effect of repeated (1,'30 s) tactile stimulation of the siphon (1000 mg) on the amplitude of the gill withdrawal reflex. -4s can be seen, when the abdominal ganglion was perfused with normal seawater )-.( the reflex habituated while 3 h later and after 20 min in picrotoxin-containi~~g seawater, the reflex no longer habit~~ated ( :-n). (H) The data are as in -4, only they have not been normalired In order t o show that picrotoxin also affected the art~plitudeof the gill withdrawal reflex in addit~onto affecting the reflex latency and habituation. In both -4 and I3, the the standard error of the mean has been plotted. mean
+
Thus, when the abdominal ganglion was perfused with picrotoxin-containing seawater the reflex latency was significantly reduced, the reflex amplitude was significantly increased, and the reflex no longer habituated with repeated siphon stimulation. Picrotoxin appeared to act specifically on those neural pathways involved in the control of the defensive gill withdrawal reflex. The neural pathways which mediate the spontaneous rhythmic gill respiratory movements were not affected by picrotoxin as evidenced by no observable changes in either the amplitude or the frequency of these contractions. Thus, the effects brought about by picrotoxin were not only reversible but were specific to the neural pathways which exert suppressive control over gill reflex behaviours. Because the picrotoxill effects are both specific and reversible they may help in the identification and characterization of the neural pathways in the CNS which exert control over the PNS. The data obtained in this present study are similar to the data obtained previously when the branchial Discussion nerve influence to the gill was removed (Lukowiak The data presented here show that the CNS's sup- 1977). In that study, following ren~oval of the pressive influence on the PNS can be manipulated branchial nerve influence, the reflex latency deby picrotoxin at concentrations of 10 6-10-W. creased, the reflex amplitude increased, and the re-
toxin to the bathing solution surrounding the ganglion the reflex no longer habituated. Both the normalized and unnormalized group data have been plotted to show better the effect of picrotoxin on the ability of the reflex to habituate. There is no doubt that the addition of picrotoxin prevents habituation of the reflex from occurring. This effect of picrotoxin was completely reversible because in a number of preparations 3 h later the reflex once again habituated. Although not shown here, the presentation of a novel stimulus was much more effective when picrotoxin-containing seawater was bathed over the ganglion. The sensitizing effect of the novel stimulus resulted in reflex amplitudes that were facilitated by up to 250% of the initial response and this facilitated state persisted for 3-5 min. This phenomenon will be examined and reported in greater detail at a later time. Thus, picrotoxin not only had the effect of potentiating the reflex a~nplitudebut also preventing habituation from occurring.
1082
CAN. J. PHYSIOL. PHARMACOL. VOL. 56. 1978
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flex no longer habituated with repeated stimulation. These changcs in gill reflex behaviour were not accompanied by any change in synaptic input or change in synaptic eficacy to the gill motor neurons in the abdominal ganglion (Lukowiak 1977). Whether this will also be the case with the picrotoxin experiments remains to be determined. Picrotoxin may bring about its effects on gill withdrawal reflex behaviours by antagonizing GABA. Recently, Tremblay and Plourde ( 1977) have shown in Aplysia central neurons that GABA's actions are antagonized by picrotoxin at concentrations similar to those used here. Further support for this notion are preliminary data which show that in young Aplysia, where the suppressive C N S control is not yet functional (Lukowiak 1978a), perfusion of GABA over tlae abdominal ganglion results in a significantly faster rate of habituation. Thus, GABA appears to allow the suppressive influence to become functional (Lukowiak: in preparation). GABA may thus be of prime importance in the neural pathways which exert suppressive control of gill reflex behaviours. Picrotoxin has previously been used to attempt to distinguish between possible neural mechanisms of habituation. Habituation could be due to active suppression or it could be due to a decrease in synaptic efficacy intrinsic to the reflex's excitatory pathways. If habituation were due to active suppression, then picrotoxin which blocks known inhibitory processes should prevent or at least retard the rate of habituation. Using this argument, Spencer ct al. (1966) ruled out a buildup of suppression to explain habituation of the flexor reflex in the spinal cat. Krasne and Roberts (1967) similarly found that picrotoxin did not alter habituation of thc escape reff ex in crayfish. They concluded that habituation was due to a depression intrinsic to the excitatory pathways of the reflex. However, Glantz (1974) found that picrotoxin couId reverse a previously habituated response and delay the onset of habituation. He suggested that an inhibitory mechanism may play an important role in the mediation of habituation. The data presented here Iend further support to the hypothesis that inhibition plays an important role in the mediation of gill withdrawal reflex habituation in Aplysia. This is not to say that the syiaaptic decrement which occurs between the central sensory neurons and the central gill rnotor neurons in the abdominal ganglion which accompanies habituation of the gill withdrawal reflex is brought about by an increase in pre- or post-synaptic inhibition. On the contrary, the evidence to date (Castellucci and Kandel 1974) suggests that this decrement is the
result of an intrinsic property of those synapses. However, it is important to recall that this synaptic decrement continues to occur even though the reflex itself doesn't undergo habituation (Lukowiak 1977, 1978h). Habituation of this reflex is not due solely to changes which occur togethcr within the integrated CNS-PNS (Lukowiak and Peretz 1977; Lukowiak 1977). It thus appears that an increase in inhibition plays an important role in the mediation of habituation by the entire integrated system. Picrotoxin actions affect the output of the increased suppressive CNS influence brought about by repetitive stimulation of the siphon. The neural pathways which initiate this suppression are not known, nor is it known where or how in the integrated system this suppression is mediated. These questions remain to be answered. C A ~ T E L L UV., C ~and ~ , KANDEL, E. K. 1974. A quanta1 analysis of the synaptic depression underlying habituation of the gill withdrawal reflex in Aplysia. Proc. Natl. Acad. Sci. U.S.A. 71, 5004-5008. GLANTZ,R. 1974. The visually evoked defensive ref ex of the crayfish: Habituation, facilitation, and the influence of picrotoxin. J. Neurobiol. 5, 263-278. JACKLET, J. W., and LUKOW~AK, K. 1975. Neural processes in habituation and seilsitization in model systems. li'rog. Neurobiol. 4, 1-56. KANDEL, E. R. 1976. Cellular basis of behavior. An introduction to behavioral neurobiology. W. H. Freeman and Co., San Francisco. KKASNE,F. B., and ROBERTS, A. 1967. Habituation of the crayfish escape response during release from inhibition induced by picrotoxin. Nature (London), 215, 769-770. LUKOWIAK, K . 1977. CNS control of the PNS-mediated gill withdrawal reflex and its habituation. Can. J. Physiol. Pharmacol. 55, 1252-1 262. --- 1 9 7 8 ~ .Development of CNS control of the siphon evoked gill withdrawal reflex in Aplysicr. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 208. -- 1978h. Induced LD activity prevents habituation of the gill withdrawal reflex evoked by siphon stimulation in rlplysia. Soc. Neurosci., in press. L U K ~ W I AK., K , and PERETZ, B. 1977. The interactions between the central and peripheral nervous systems in the mediation of gill withdrawal reflex behaviour in Aplysia. J. Comp. Physiol. 117, 219-244. PEKETZ,R., and HOWIESON, D. 1973. Central influence on peripherally mediated habituation of an Aplysicr gill withdrawal response. J. Comp. Physiol. 84, 1-18. PPKETZ,B., and LUKOWIAK, K. 1975. Age-dependent CNS control of the habituating gill withdrawal reflex and of correlated activity in identified neurons in Apl~lsia.J. Comp. Physiol. 103, 1-17. SPENCER, W. A., THO~.IPSON, K. F., and NEILSON,I). 1966. Decrement of ventral root electrotonus and intracellularly recorded post-synaptic potentials produced by iterated cutaneous afferent volleys. J. Neurophy siol. 29, 253-274. TREMBLAY, J. P., and PLOURDE, G. 1977. Presynaptic modulatory effects of GABA on depression, facilitation, and posttetonic potentiation of a cholinergic synapse in Apljqsicr culijornicu. Can. J. Physiol. Pharmacol. 55, 1286-1 301.
