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Modulation of Gill Withdrawal Reflex Habituation in Aplysia KEN LUKOWIAK*

Department

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Physiology, McGill University, Montrcwl, Quebec, Canada

Received July 1 0 , 1978; revised December 18, 1978

SIJMMARY

Repeated tactile stimulation of the siphon in Aplysia normally results in habituation of the gill withdrawal reflex and a concomitant decrease in the amplitude of the excitatory synaptic input to gill motor neurons in the abdominal ganglion. It was found, however, that induced low-level tonic activity in motor neuron Lg, which does not itself elicit a gill withdrawal movement, prevented habituation of the reflex from occurring. Further, in preparations already habituated, this tonic low-level activity brought about a reversal of habituation. Although tonic L g activity prevented the occurrence of habituation or brought about its reversal, it did not interfere with the synaptic decremental process which normally accompanies gill reflex habituation. Motor neurons L7 and LDGl were found not to possess this ability of Lg to modulate gill reflex habituation. Evidence suggests that Lg’s modulatory effect is mediated in the periphery, in the gill and not centrally in the abdominal ganglion. INTRODUCTION

The gill withdrawal reflex and its subsequent habituation evoked by repeated tactile stimulation of the siphon in Aplysia has been extensively studied in an attempt to gain an understanding of the neural mechanisms which underlie adaptive behavior (Jacklet and Lukowiak, 1975; Kandel, 1976). It was initially proposed that both the reflex and its habituation were mediated exclusively by neural processes within the central nervous system (CNS), the abdominal ganglion (Kupfermann et al., 1970; Castellucci et al., 1970; Kupfermann e t al., 1971; Kupfermann et al., 1974; Castellucci and Kandel, 1974). However, it has been shown (Peretz et al., 1976) that there was also a peripheral pathway (the PNS) between the siphon and gill which was competent to mediate the reflex and its habituation evoked by the same siphon stimulation in the absence of the CNS. Lukowiak and Peretz (1977) further found that the CNS and PNS interacted and were parts of an integrated system which normally mediated adaptive gill reflex behaviors. The systems did not act independently. Finally, Lukowiak (1977a) examined the role played by each part of the integrated system in the * Present address: Division of Medical Physiology, Faculty of Medicine, {Jniversity of Calgary, Calgary, Alberta T2N 1N4, Canada Journal of Neurobiology, Vol. X, No. 3 , pp. 255-271 (1979) 0 1979 John Wiley & Sons, Inc.

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mediation of adaptive gill reflex behaviors. He found that the CNS exerted both facilitatory and suppressive control over neurons in the PNS which mediated the reflex and its habituation. These facilitatory and suppressive control neurons in the CNS were not identified. In a recent series of experiments (Lukowiak, 197713; 1979) it was found that Lg, a central gill motor neuron, was a modulator neuron. Induced Lg activity significantly potentiated L7’s ability to elicit a gill withdrawal response. Since induced activity in Lg exerted modulatory control on the gill withdrawal response elicited by L7 activation, I examined whether induced Lg activity might also exert modulatory control over the gill withdrawal reflex and its habituation evoked by siphon stimulation. Because Lukowiak and Peretz (1977) also found th a t induced low-level activity in L7 modulated the amplitude of the gill withdrawal reflex evoked by siphon or gill stimulation, I also examined whether such activity in L7 or LDGl would exert modulatory control over habituation of the reflex. I now report that induced low-level Lg activity, which does not itself elicit a gill movement, prevented habituation of the gill withdrawal reflex or resulted in its reversal in an already habituated preparation. Induced activity in L7 or LDGl did not affect habituation. The induced Lg activity did not affect the synaptic decrement which normally occurred between central mechanoreceptor neurons and central gill motor neurons during habituation. Lg’s modulatory control is probably mediated peripherally in the gill. METHODS Twenty Aplysia californica, obtained from Pacific Riomarine Laboratories (Venice, CA), weighing between 150-250 g were used. The animals were maintained in artificial sea water (Instant Ocean) at 15-16OC and pH 7.9. The preparation used in this study was similar to that previously described (Peretz et al., 1976; Lukowiak, 1977a) and is shown in Figure 1. I t consisted of the siphon, mantle, gill, and abdominal ganglion. The branchial, ctenidial, and siphon nerves by which the abdominal ganglion innervates the siphon and gill were left intact, all other nerves were severed. The preparation was kept a t 15-16°C in a 1000-ml chamber of artificial sea water during the course of the experiment. The gill withdrawal reflex amplitude was measured using a Grass Force Transducer (FT 03) connected to a single gill pinnule by fine surgical thread. The output of the transducer was recorded on a storage oscilloscope and a polygraph from which the measurements were made (7 mm = 200 mg). Recording gill movements in this manner did not result in any observable damage to the gill and preparations remained viable for a t least 24 hr. The criterion used to determine if the preparation was viable was the appearance of periodic spontaneous gill respiratory movements. If a preparation did not exhibit these gill movements during the first hour or so following surgery, it was discarded. Data were obtained and used from all preparations which exhibited these spontaneous contractions. Data were not excluded because of small amplitude reflexes (Carew et al., 1976), but in all cases the reflex amplitudes were a t least 25% of the magnitude of the large gill respiratory movements. The amplitude of these spontaneous gill respiratory movements, which are attributed to activity of the interneuron I1 network (Kupfermann e t al., 1974). appeared to be maximal amplitude gill contractions; since the amplitude of these contractions were similar to the contractions evoked in a number of the preparations by cutting of the brancbial and ctenidial nerves. The absolute magnitude of these large spontaneous gill movements in the preparations used ranged between 600 and 2000 mg (see C in Fig. 1). Tactile stimuli were delivered to the siphon by a “tapper” which is a plastic coated wire, 1 mm in diameter, which was connected to a solenoid (see Peretz and Lukowiak, 1975). The duration and amplitude of the voltage impressed across the solenoid coil and the distance from the stimulation

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site (normally less than 1mm) determined the force applied. The stimulus intensity used in these experiments was 1000 mg with a duration of 30 msec. This can be considered as a weak to moderate intensity stimulus (Peretz et al., 1976). All experiments reported here used the “tapper” to evoke the reflex. It needs to be noted that the stimulation used by Byrne et al. (1974) did not evoke a gill withdrawal reflex after removal of the abdominal ganglion whereas the “tapper” did. The major differences between the stimulators were the duration and the type of stimulus produced. The stimulus employed by Byrne had a duration of 800 msec and produced a constant force for that time. It is not understood why one type of stimulator can activate the PN S and the other not, when they both activate the CNS in much the same manner. Neuron identification was consistent with that of Koester and Kandel (1977) and was based on a number of criteria. For neurons L7 and LDGl the criteria included: soma location; size; synaptic activity during periodic spontaneous gill respiratory movements; the appearance of one-for-one activity recorded in a gill pinnule by an extracellular suction electrode (referred to as a pinnule potential) with the intracellularly recorded action potential (AP); the type of gill movement elicited by its depolarization. The pinnule potentials may be an extracellular recording of the E J P in gill muscle cells (see Jacklet and Rine, 1977). Similar criteria were also used to identify Lg. However, Lg must be depolarized to produce a higher rate of AP’s for a longer duration than L7 to produce the same amplitude of contraction. One-for-one pinnule potentials were never observed with Lg activity and Lg innervates the gill via a small branch of the siphon nerve and not via the branchial or ctenidial nerves. In this regard Lg is unique. There are a t least two Lg motor neurons in each abdominal ganglion, each of which causes a similar contraction. Normally the larger of the two was recorded from. There did not seem to be any qualitative or quantitative difference between the Lg’s with respect to their effect on gill withdrawal reflex habituation. Micropipetes filled with 3 M KC1 and having a resistance of 15-30 M were used. A bridge circuit in the electrometer (Getting M-5) allowed simultaneous recording and stimulation. Constant current was applied intracellularly to the neuron to produce a steady depolarization which resulted in AP’s. The intensity of the current was adjusted to produce the desired rate of AP’s (1-5 AP’s/sec). The rate of AP’s used in each cell was adjusted so that an observable gill movement was not observed. An habituation series consisted of a t least 10 presentations of the tactile stimulus to the siphon with an interstimulus interval (ISI) of 30 sec. Each series was followed by a rest period of a t least 3 hr (see Peretz and Howieson, 1973) and each preparation was normally tested four times: two series with Lg depolarized to produce 1-5 AP’s/sec and two with Lg free running. Lg’s spontaneous activity is very similar to that of gill motor neuron L7 and exhibits an irregular firing pattern (Frazier et al., 1967). Lg is most active just following a spontaneous gill respiratory movement (Fig. 1C) where it can reach a firing frequency of up to 5-6 AP’s/sec. Lg was never observed to be tonically active. Normally, during a habituation run the large spontaneous gill respiratory movements are not often observed. In most preparations, L7 or LDGl was also recorded from during the four series. In some control experiments L7 or LDGl was depolarized during a habituation series to determine the effect, if any, on gill reflex habituation. Where normalized data were presented the initial response was taken as 100% and all other responses as a percentage of that. In grouped data the mean and standard error of the mean are plotted.

RESULTS

I t was known from previous work (Lukowiak and Peretz, 1977) that induced low-level activity in L7 (1-3 AP’s/sec) modulated the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon. While this induced activity itself did not result in an observable gill movement, the reflex amplitude evoked by siphon stimulation was larger than when L7 was free running. Results similar to these have now been obtained with LDGl (Lukowiak, in preparation). Since induced low-level activity in these gill motor neurons resulted in modulation of the amplitude of the gill withdrawal reflex, it seemed plausible that such

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Fig. 1. (A) The abdominal ganglion (CNS) innervates the gill via the Br (hranchial), Ct (ctenidial), and a small branch of the Sn (siphon nerve, not shown). The abdominal ganglion innervates the siphon via Sn. The GG (gill ganglion) is located on the Br in the musculature ofthe efferent vessel of the gill. Punctuate tactile stimuli were delivered to the siphon (indicated by the asterik) by a mechanical tapper. The gill withdrawal reflex evoked by tactile stimulation involved the whole gill. T r (surgical thread) was attached to a single gill pinnule and led to a force transducer to record gill movements (in some experiments, siphon movements were also monitored by similar means). The abdominal ganglion was pinned out on a clear sylgard platform and transilluminated which aided in the impalement of neurons such as L7. (B) Schematic diagram of the known and hypothesized connections of the identified neurons in this study. Sensory information (SEN) is conveyed from the siphon to motor neurons L7 and Lg in the central nervous system (CNS) via the siphon nerve to sensory neurons (Byrne et al., 1974). Sensory information is also sent to peripheral motor neurons (PMN) in the peripheral nervous system (PNS)via a peripheral pathway between the siphon and gill (Peretz et al., 1976; Lukowiak, 1977a), Li synapses directly onto muscle fibers in the gill (Carew et al., 1974;Jacklet and Rine, 1977) and the extracellulary record pinnule potentials reflect the EJI's recorded in these fihers. Pinnule potentials have not as yet been correlated with L g activity but have with the other gill motor neurons, thus, it is not known whether Ly synapses onto gill muscle (GM) or brings about its contraction via a PMN. Lg, however, has been shown to be dopaminergic (Swann et al., 1978a, b) and, based on the evidence obtained here and with dopamine perfusion through the gill (Ruben and Lukowiak, 1979), five possible sites where I+ could have its affect on habituation have been proposed (see text for details). (C) A spontaneous gill respiratory movement (middle trace) which is attributable to the interneuron I1 network in the abdominal ganglion is shown. Note that Lg (lower trace) is initially inhibited during the gill movement and then exhibits rebound excitation. Motor neuron I,7 behaves in a very similar manner. The upper trace is an extracellular recording made from the gill. One for one pinnule potentials were never observed with Ly activity hut were observed with activity in other gill motor neurons. The spontaneous contraction is a near maximal gill contraction. These data were obtained from the same preparation shown in Figure 7 but before L7 was impaled. Scales 40 mV (1,s); 200 mg ( G i ) ;20 ~ L (pinnule V potential); 10 sec.

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induced activity might also modulate habituation of the reflex. Eleven preparations were tested to determine if induced low-level activity in these cells effected gill reflex habituation according to the protocol described in the methods. I t was found that induced activity in L7 or LDGl did not modulate habituation of the gill withdrawal reflex; habituation was similar with and without the induced activity in these neurons. These data are presented in Figure 2A (L7) and 2B (LDG1). The induced activity did, however, affect the amplitude of the reflex (Fig. 3A and 3B) but it had no effect on the rate or degree of habituation. Because it had previously been shown (Lukowiak, 197713; 1979) th a t Lg was

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Fig. 2. Effects of induced low-level tonic activity in gill motor neurons L7 and LDGI. (A) In 11 preparations with L7 free running, repeated tactile stimulation of the siphon (1000 mg, IS1 30 sec) resulted in habituation of the gill withdrawal reflex ( I - = ) In . each preparation, after a rest of 3 hr, L7 was depolarized to produce 1-3 AP’s/sec and the siphon was again stimulated as before. As can be seen ( 0 . ..O) this again resulted in habituation of the reflex. (B) In 11preparations LDGl was tested as described in (A). With LDGl free running (m-m) and with induced tonic activity in LDGl (0...O) repeated tactile stimulation of the siphon resulted in habituation. The mean f S.E.M. have been plotted.

a modulator of Ly’s, and to a lesser extent of LDGl’s, ability to elicit a gill withdrawal response, it seemed appropriate to determine if induced low-level tonic L g activity could modulate gill withdrawal reflex habituation. Thus 15 preparations were tested according to the protocol described in the Methods section. The pooled results from these 15 preparations are shown in Figure 4. As can be readily seen, induced tonic activity in L g prevented habituation (0. - 0 ) . When L g was free running (m-.) repeated siphon stimulation resulted in the expected gill withdrawal reflex habituation. Note also that the induced Lg activity also modulated the amplitude of the evoked reflex in much the same manner as L7 or LDGl did. Thus induced low-level tonic activity in Lg modulated both the reflex amplitude and habituation of the reflex evoked by tactile stimulation of the siphon. I t was possible that the prevention of gill reflex habituation by the induced tonic Lg activity could have been the result of a failure of the synaptic input from the central mechanoreceptor neurons to the central gill motor neurons in the abdominal ganglion to decrement as normally occurs with repeated tactile stimulation (Kupfermann et al., 1970;Lukowiak, 1977a). T o test this possibility, L7 was recorded from both when Lg was free running and when tonic activity was induced in it. Data from two such experiments are shown (Fig. 5). In each, the amplitude of the gill withdrawal reflex and the number of AP’s evoked on each trial in L7 by the tactile stimulus are plotted. As can be seen when L g was free running, repeated siphon stimulation resulted in habituation of the gill withdrawal reflex).-.( and a concomitant decrease in the number of AP’s evoked on each trial in L 7 (0--0). However, 3 hr later, with induced tonic Lg activity, repeated siphon stimulation no longer resulted in habituation of the gill withdrawal reflex (A-A), but the number of AP’s evoked in L7 on each trial con-

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Fig. 3. Effect of induced low-level tonic L7 and LDGl activity on the amplitude and habituation of the gill withdrawal reflex. (A) In a single preparation with L7 free running (m-m) repeated tactile stimulation of the siphon (1000mg, IS1 30 sec) resulted in habituation of the gill withdrawal reflex. Three hours later and with L7 depolarized to produce 1-3 AP’shec, the gill withdrawal reflex evoked by the same tactile stimulus is larger (0.. .O)but repeated siphon stimulation continues to result in habituation. (B) As in (A), only LDGl was used. Again, with induced tonic LUG1 activity (1-2 AP’shec) the gill withdrawal reflex was larger (0.. - 0 )than when LDGl was free running (m-m) but habituation still occurred.

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tinued to decrement as in the initial run ( 0 - - 0 ) . Thus the synaptic input from the central mechanoreceptor neurons to the central gill motor neurons, as evidenced by the number of AP’s evoked on each trial in L7, continued to decrement even though Lg activity prevented habituation of the reflex. Therefore Lg’s modulatory effect on habituation is not the result of an interference with the decremental process which occurs a t these central synapses. Results similar to those presented in Figure 5 were obtained in four other preparations tested in a like manner. In all cases tested, there was never any increase in the number of AP’s evoked in L7 with repeated stimulation while low-level tonic activity was induced in Lg. In addition to Lg’s ability to prevent or retard habituation, it was found that induced low-level tonic activity in Lg could bring about the reversal of habituation in a preparation already habituated. This can be seen in Figure 6. In six preparations tested, the gill withdrawal reflex was first habituated by repeated tactile stimulation of the siphon with Lg free running (Fig. 6A, trials 1-10). Following the 10th trial, L g was depolarized to produce 3-5 AP’s per second and as can be seen (trials 11-20) the amplitude of the gill withdrawal reflex became larger with repeated stimulation. Thus by trial 20, it approached the initial reflex amplitude. As soon as Lg was again allowed to free run (following trial 20) the reflex amplitude once again exhibited its expected decrement. In Figure 6B,

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Fig. 4. Induced low-level tonic activity in L g effects habituation of the gill withdrawal reflex evoked by tactile stimulation of the siphon. Pooled data (N = 15; f S.E.M.) showing the gill withdrawal reflex evoked by repeated tactile stimulation of the siphon (1000 mg, IS1 30 sec) with Lg free running (I-=) and with L y depolarized to produce 1-5 AP’s/sec ( 0 -. - 0 ) . As can be seen with L g free running, the reflex habituated. Three hours later, in each preparation with induced tonic activity in L g , the produce 1-5 AP’s/sec did not result in an observable gill withdrawal movement.

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the results from a similar experiment are shown. Initially, low-level tonic activity in Lg prevented habituation (0 0).When Lg was allowed to free run the amplitude of the reflex habituated ( A-A) but as soon as low-level tonic activity was induced again in L g the reflex amplitude grew with repeated stimulation. Results similar to those presented in Figure 6A and 6B were obtained in four other preparations and the polygraph data from one of these preparations are presented in Figure 7. Again, the reflex amplitude decremented until such time as low-level tonic activity was induced in Lg (trials lo-20), then the reflex amplitude grew with each stimulus presentation. Finally, when Lg was again allowed to free run, the reflex amplitude again decremented with repeated siphon stimulation. As can also be seen, there was no concomitant increase in the number of AP’s evoked in L7 by the siphon stimulation when low-level tonic ac-a-

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Fig. 5. Induced tonic activity in L g , while it prevented habituation of the reflex, it did not interfere with the synaptic decremental process between central sensory neurons and central gill motor neurons which accompanies gill reflex habituation. (A) In a single preparation with Lg free running, the siphon was stimulated (1000 mg, IS1 30 sec) and the gill reflex amplitude and the number of AP’s evoked in L 7 on each trial were recorded. As can be seen, the gill withdrawal reflex habituated (.-a) and the number of AP’s evoked on each trial decremented a t a similar rate (0.. -0). After a 3-hr rest ( f ) Lg was depolarized to produce 1-5 AP’s/sec. Now repeated siphon stimulation did not produce habituation of the reflex (A-A), however, the number of AP’s evoked in L7 on each trial continued to decrement as before (0.. - 0 ) . (B) In another preparation, the same experiment as described in (A) was performed. Again, with induced Lg activity (A-A), the reflex did not ha-0). bituate, but the number of AP’s evoked in L7 continued to decrement (0..

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Fig. 6. Induced tonic L g activity reverses habituation of the gill withdrawal reflex. (A) With Lg free running (m-m), repeated tactile stimulation of the siphon (1000 mg; IS1 30 sec) resulted in habituation of the reflex. Following trial 10, Lg was depolarized to produce 3-5 AP’s/sec. As can be seen (0.. .o), with induced tonic Lg activity, the reflex amplitude increased with repeated tactile stimulation so that by trial 20 it approached its initial amplitude. Following the 20th trial, the depolarizing current was shut off (m-m) and the reflex amplitude immediately decreased and then exhibited decrement with further stimulation. (B) In another preparation with L g free running, repeated siphon stimulation resulted in habituation of the reflex (.-=I. After a 3-hr rest and with induced tonic activity in L g (0.. - 0 )repeated siphon stimulation no longer resulted in habituation. After the 10th trial L y was allowed to free run (A-A) and as can be seen, the reflex now habituated. When Lg was again depolarized to produce 3-5 AP’s/sec (followingtrial 16) the reflex amplitude became larger with repeated stimulation ( 0 . . .O).

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tivity was induced in Lg. Thus induced Lg activity cannot only prevent the occurrence of habituation, but it can bring about its reversal. However, induced tonic Lg activity did not interfere with or bring about a reversal of the synaptic decrement associated with gill withdrawal reflex habituation. DISCUSSION

The data shown in Figure 4 clearly indicated that induced low-level tonic activity in L g prevented habituation of the gill withdrawal reflex. In the 15 preparations tested, it was first demonstrated that repeated tactile stimulation of the siphon resulted in habituation of the reflex. The habituation curve obtained from these 15 preparations is quite representative of gill withdrawal reflex habituation curves presented elsewhere (Peretz et al., 1976; Kandel, 1976; Lukowiak, 1977). Thus, these 15 preparations responded normally to repeated tactile stimuli applied to the siphon. After a 3-hr rest the preparations were again stimulated similarly. However, during this stimulus series, Lg was depolarized to produce 1-5 AP’sIsec. This induced activity in Lg prevented habituation of the gill withdrawal reflex. In some of the preparations, the amplitude of the gill withdrawal reflex increased up to 150%of its initial amplitude with repeated stimulation. Other gill motor neurons were tested in a similar manner to determine if induced tonic activity could also prevent habituation. Induced tonic activity in Lg and LDGl did not prevent habituation (Figs. 2 and 3). Thus the modulatory control property of Lg over habituation appears to be a specific property of Lg and not of motor neurons in general. While induced tonic activity in L7 or LDGl did not prevent habituation of the reflex, the induced activity did modulate the amplitude of the reflex. With induced tonic low-level activity in these neurons, the amplitude of the reflex was

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Fig. 7. Data taken from polygraph records from another preparation tested as in Fig. 6. The data are from trials 1,5,10,11,1~3,20,23, and 25. Along with the gill reflex amplitude the response evoked by the stimulus in cells L7 (top) and Lg (bottom) are shown. Lg was free running (trials 1-10) and the gill reflex habituated and the number of AP's evoked in L7 also decremented. Following the 10th trial induced Lg activity brought about the reversal of habituation (trials 11-20). Notice, however, that there was no large increase in the number of' AP's evoked in L7 even though the amplitude of the reflex grew. Following trial 20, Lg was again free running and the reflex again habituated. Scales: 1 sec; L7, 30 mV; Lg, 40 mV.

larger than when the neuron was allowed to free run (Fig. 3 ) . Similar results were also obtained with induced tonic activity in L g (Fig. 4). These findings are in general agreement with those reported by Lukowiak and Peretz (1979) and a more thorough report and analyses of these data is in preparation. Ly activity also modulated the effectiveness of L7 to elicit a gill withdrawal movement (Lukowiak, 197713; 1979). Following Ly activity, L7's ability to cause a gill withdrawal movement was clearly potentiated. Sinback (1975) also found that

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induced tonic activity in L7 and LDGl together modulated the periodicity of the centrally controlled gill respiratory movements. It thus appears that in addition to their function as gill motor neurons, L7, LDGI, and in particular Lg, possess different modulatory functions. Thus neurons in this relatively simple system have a multiplicity of function (see also Weiss et al., 1978). The particular function brought into play may depend on the particular frequency of activity of the cell or when in relation to another cell’s pattern of activity it is active. Induced tonic activity in Lg did more than just prevent habituation of the reflex, it could also bring about the reversal of habituation in a preparation already habituated (Figs. 6 and 7). The reversal of habituation did not occur immediately after the onset of tonic activity in Lg; it took a t least 7 trials before the amplitude of the reflex reached its initial size. Since there was a steady increase in the amplitude of the reflex with further tactile stimulation the effect was not just due to the fact that Lg modulates the amplitude of the reflex. Because if that were the case, it would be expected that after the initial increase in size of the reflex the amplitude of the reflex would decrement with further stimulation just as was observed with induced tonic activity in L7 or LDGl (Figs. 2 and 3). Nor was the tonic induced activity in Lg dishabituating the reflex. Peretz and Lukowiak (1975) showed that the interposition of L7 or LDGl activity served as a dishabituatory stimulus; however, the effect was only transitory, lasting only 1or 2 trials and when repeated, its dishabituatory effect diminished. This was not the case with the six preparations tested. As long as tonic activity was maintained the amplitude of the reflex increased until it reached its initial level, after that the amplitude did not vary to any great extent with further stimulation. This effect of a reversal of habituation, however, was not persistent. Within one or two trials after the cessation of induced tonic activity the reflex once again began to habituate. While induced low-level tonic Lg activity prevented habituation or brought about its reversal it did not interfere in any observable way with the synaptic decremental process which occurs between the central mechanoreceptor neurons and the central gill motor neurons in the abdominal ganglion as a result of repeated tactile stimulation of the siphon. As was shown (Fig. 5) with Lg free running the number of AP’s evoked on each trial in L7 decremented as did the amplitude of the gill withdrawal reflex with repeated siphon stimulation. The data shown here for the decrement in the number of AP’s evoked in L7 are very similar to those shown by Kupfermann et al. (1970, Fig. 2A) and strongly argue that the stimulus used here had quite a similar effect as the 500-msec jet of seawater used in their earlier study. In any case, 3 hr later, with induced low-level tonic Lg activity the reflex no longer habituated, but the synaptic input to L7 from the central sensory neurons, as evidenced by the number of AP’s evoked in L7 on each trial (Kupfermann et al., 1970), continued to decrement as before. Thus Lg’s modulatory effect on habituation did not involve an alteration of the synaptic decremental process which normally accompanies habituation. Similarly, the reversal of habituation brought about by the introduction of induced tonic Lg activity did also not involve an alteration of the synaptic decremental process (Fig. 7). Thus, Lg must be acting elsewhere.

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The fact that induced tonic Lg activity did not alter the synaptic decremental process in the abdominal ganglion while it prevented habituation of the reflex is similar to the results obtained following branchial nerve cut (Lukowiak, 1977a). Following cutting of the branchial nerve (the siphon and ctenidial nerves were still intact) repeated tactile stimulation of the siphon (1000 mg, IS1 30 sec) resulted in response sensitization even though the number of AP’s evoked per trial in L7 or LDGl continued to decrement as before when the branchial nerve was intact and repeated siphon stimulation resulted in habituation. Lukowiak (1977a) concluded on the basis of those and other results that the CNS exerted facilitatory and suppressive control over the P N S mediated behaviors. Thus, when the branchial nerve was severed, which removed the CNS’s suppressive control over the PNS, the facilitatory control dominated (mediated via the ctenidial nerve) and sensitization resulted. It may thus be that L g can act as one of the central facilitatory control neurons in the mediation of gill reflex behaviors. In addition to the fact that induced tonic Lg activity did not alter the synaptic decremental process, a number of other lines of evidence suggest that Lg’s effect on gill reflex is mediated in the periphery, in the gill itself. Lukowiak (197713; 1979) found that Lg’s modulatory effect on L7’s ability to elicit a gill withdrawal response was mediated peripherally in the gill and not in the abdominal ganglion. Secondly, although Lg has not definitely been shown to be dopaminergic, the evidence gathered to date strongly suggests that it is (Swann e t al., 1978a, b). I t was found that the infusion of dopamine through the gill potentiated L7’s ability to elicit a gill withdrawal response in much the same manner as induced Lg activity did (Lukowiak, 197713; 1979). Further, in a series of experiments (Ruben and Lukowiak, 1979) the infusion of dopamine through the gill resulted in an increased gill reflex amplitude and the prevention or retardation of gill reflex habituation; again just as induced tonic Lg activity did. These experiments were all performed with the abdominal ganglion isolated so th a t the perfusate containing dopamine could not come into contact with the central neurons. However, where in the gill the effect is mediated is not known. Based on the available evidence, five possible sites where Lg could modulate the reflex and its habituation have been proposed (Fig. 1B). Lg could presynaptically facilitate the neuromuscular junction of the central efferents such as L7 (site 1). Evidence to support this has been obtained (Swann et al., 1978b) which show th a t both the amplitude of the extracellularly recorded pinnule potential which showed one for one activity with L7 and the amplitude of the gill response elicited by L7 activity was greatly facilitated. Lg could bring about its effect by directly acting on gill muscle so that any input to the muscle would be much more effective (site 2). Ruben (1977) found that dopamine does have a direct affect on gill muscle which can lead to a facilitated response. Lg (site 3 ) could also modulate habituation by presynaptically facilitating the neuromuscular junction of peripheral motor neurons (PMN) (Peretz and Estes, 1974) which mediate the basic reflex and its habituation (Lukowiak, 1977a). Presently, there is no evidence for or against this proposal. However, experiments in progress in which either the CNS or P N S pathway from the siphon to the gill have been removed may help to

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differentiate between sites 1 and 3. If Lg does not synapse directly onto gill muscle but rather works via a PMN (site 4)then low-level induced Lg activity may lead to facilitation of that synapse or the PMN junction with the muscle. Thus any other input to the PMN (e.g., via SEN) would lead to an increased reflex amplitude or a nonhabituating reflex. Finally (site 51, Lg could presynaptically facilitate the SEN input to the PMN. Again, there are no present data to support this notion. However, the experiments mentioned above may help to distinguish between this possibility. Whether the modulatory affect of Lg on habituation involves a change in cyclic AMP levels as has been proposed to account for sensitization in the abdominal ganglion (Brunelli et al., 1976) and which may mediate the metacerebral cells’ modulatory affect on buccal muscle (Weiss et al., 1978) needs to be determined. There is evidence (Kebabian et al., 1977) that the infusion of dopamine into the gill results in an increase in cyclic AMP levels, but whether such increases in cyclic AMP levels account for Lg’s modulation of habituation remain to be determined. A question that arises is whether Lg is ever tonically active, as was artifically induced in these experiments and so prevent habituation of the gill withdrawal reflex. Under the stimulus conditions normally used by experimenters Lg exhibits an irregular lining pattern much like L7 and the answer would appear to be no. Stimulus intensities and ISI’s are picked so as to produce habituation. The parameters chosen do not lead to tonic activity in Lg. However, with a much stronger stimulus applied to the siphon Lg does fire tonically and the rate of habituation is much less or habituation does not result (unpublished observations). Also after the large spontaneously occurring gill respiratory movements which occasionally occur during a habituation series, the amplitude of the next gill withdrawal reflex is larger and may stay larger for a few trials. During the spontaneous contraction Lg is hyperpolarized and following this hyperpolarization there is a rebound effect and Lg is tonically active, 3-5 AP’s/sec for a minute or so. This spontaneously occurring tonic Lg activity may explain the increase in gill withdrawal reflex amplitude which is similar to that seen during the reversal of habituation (Figs. 6 and 7). Habituation of the gill withdrawal reflex evoked by tactile stimulation of the siphon is brought about by changes in the integrated CNS-PNS (Lukowiak, 1977). The data presented here again show that habituation cannot be mediated solely by changes in synaptic efficacy which occur between central sensory neurons and central gill motor neurons in the abdominal ganglion. These changes are no doubt important and may constitute the best model system to date for the study of synaptic changes associated with behavioral modifications, but these changes cannot alone constitute the neural mechanisms of gill reflex habituation. If these changes did constitute the neural basis of gill reflex habituation then Lg activity of necessity would have had to affect the synaptic decremental process, which of course, it did not. To fully understand the neural basis of gill withdrawal reflex habituation it is necessary to take the complete integrated CNS-PNS system into consideration and determine where and how the neural changes are occurring. This work was supported by the MRC of Canada.

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