JOURNAL OF NEUROBIOLOGY, VOL.
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HABITUATION AND D ISHAB ITUATI ON MEDIATED BY THE PERIPHERAL AND CENTRAL NEURAL CIRCUITS O F THE SIPHON OF APLYSIA K E N N E T H LUKOWIAK and JON JACKLET Department of Biological Sciences and Neurobiology Research Center, State University of New York at Albany, Albany, New York 12222
SUMMARY
The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Responses evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems. INTRODUCTION
Habituation, perhaps the most elementary and ubiquitous form of behavioral plasticity, has been defined as a decrease in response to repeated stimulation (Harris, 1943). More explicitly, habituation in invertebrate preparations has been operationally defined by a set of 9 parametric characteristics (Thompson and Spencer, 1966). The three characteristics that are generally sufficient to establish that a given response habituates are: 1)exponential decrease in response strength to repeated stimulation, 2) spontaneous recovery, and 3 ) dishabituation (Eisenstein and Peretz, 1973). Habituation of spinal reflexes has been shown to occur in the absence of higher neural centers (Prosser and Hunter, 1936) in polysynaptic pathways of the spinal cord (reviewed by Thompson and Spencer, 1966; Griffin, 1970; Thompson, Patterson and Teyler, 1972). In the invertebrate Aplysia, habituation of siphon and gill withdrawal has been at183 @ 1975 by John Wiley & Sons, Inc.
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tributed to changes in the activity of central neurons (Castellucci, Pinsker, Kupfermann and Kandel, 1970; Kupfermann, Castellucci, Pinsker and Kandel, 1970; Pinsker, Kupfermann, Castellucci and Kandel, 1970). However, the responses (gill and siphon withdrawal) that were examined have been shown to undergo habituation in the absence of central neurons (Peretz, 1970, Lukowiak and Jacklet, 1972). These results raise the question of how much each system, central and peripheral, contributes to the withdrawal responses. We examined the characteristics of habituation and dishabituation in the isolated siphon, the contribution of the central neurons to the siphon response, and the modulatory influences between the peripheral and central circuits. METHODS
Sixty Aplysia californica, obtained from Pacific-Bio-Marine (Venice, Calif.) were used in these experiments. The animals were maintained prior to use in artificial seawater (Instant Ocean) a t 15-17°C under a light-dark cycle of 13 hr light and 11 hr dark. I n preparation the animal was pinned out dorsal side up on a tray and a n incision was made just rostra1 to the mantle. T h e pleural-abdominal connectives were cut and the siphon, mantle, gill, and abdominal ganglion complex were cut free from the animal. This complex was reduced to the two preparations (Fig. I) used in this study: (I) the siphon-mantle connected to the abdominal ganglion by the siphon nerve only (the semiisolated preparation) and (2) the siphon without the abdominal ganglion (the isolated preparation). The preparations were pinned out completely submerged under artificial seawater (19-22°C) in a chamber with a Sylgard 184 (Dow-Corning, Midland, Mich.) Retractor Abdominal Ganglion
Fig. 1. Diagrammatic representation of the semi-isolated preparation. The abdominal ganglion is attached to the siphon, mantle, and gill by the siphon nerve. EXtracellular recordings were made from the siphon nerve and intracellular recordings were made from neurons in the abdominal ganglion. The siphon nerve was stimulated electrically (n).The nerve was severed at the arrow to make a n isolated preparation. S.N., siphon nerve; L.C. and R.C., left and right connectives; B.N., branchial nerve.
(4)
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base. A thread was attached from the siphon to a Grass Tension Transducer (Ft 03) and withdrawal was measured as a voltage deflection (60 mV = 1 g) on either a polygraph or a storage oscilloscope. Neuronal activity in the siphon nerve was monitored by extracellular recordings with suction electrodes (made en passant in the semi-isolated preparations) or electrical stimulation with 1 msec square pulses was delivered through the same electrodes. Light and tactile stimuli were used to evoke the withdrawal response of the siphon. The light stimulus was approximately 1000 lumen,/mZand 6 sec in duration from a n incandescent lamp. The tactile stimulus was a single drop of seawater falling from a height of 5 cm. The stimuli were presented with an interstimulus interval (ISI), usually of 30 sec. The preparations were set up in the chamber and then allowed to rest for a t least 45 min before testing was begun. I n some experiments isotonic sucrose (0.8 M ) was used to block axonal conduction in the siphon nerve in order to isolate the central neurons in the abdominal ganglion from the effects of peripheral stimulation. Isotonic sucrose solution was flowed over the siphon nerve from polyethylene tubing a t a slow rate and was drawn off by another tube. The sucrose displaced the ions necessary for the propagation of the action potential and caused a block that was confirmed by extracellular recordings from the siphon nerve. The block was relieved within 30 sec after cessation of the sucrose flow. RESULTS
Siphon withdrawal responses The semi-isolated and isolated siphon preparations respond to tactile or light stimulation. The response consisted of longitudinal contraction of the siphon, so that the tip of the siphon is withdrawn toward the mantle, and a concomitant constriction of the siphon lumen. I n these experiments we measured the tension developed by longitudinal contraction. The tension evoked by a tactile stimulus in an isolated preparation is shown in Figure 2A. The stimultaneous extracellular recording from the siphon nerve indicates that afferent activity was evoked by the stimulus and occurred with a latency similar to that for tension. This afferent activity would presumably activate centra! neurons. Figure 2 B shows the response evoked by light stimulation of an isolated preparation. Note that the extracellular recording from the siphon nerve revealed very little activity associated with the light stimulus. This has been a consistent finding. The duration and amplitude of the responses depends upon the particular preparation and the history of stimulation; however, in the rested condition the tension was about 3-5 g for light or tactile stimulation. A comparison was made between the latency of the siphon contraction to the tactile stimulus in the semi-isolated and the isolated preparations. The mean latency in the isolated preparations ( n = 36, 18 preparations) was 118 msec (S.D. = 5.9) while the mean latency in the semi-isolated preparations ( n = 40, 32 preparations) was 119 msec (S.D. = 4.2). Thus, there is no difference in the latency. Also the response amplitudes evoked by tactile stimulation were compared in the same preparation with and without the abdominal ganglion. The response of the semiisolated preparation was measured after a 1hr rest and again 1hr after the
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Fig. 2. Comparison of the latency of the siphon contraction and evoked neural activity in isolated preparations to tactile and light stimulation. (A) Siphon tension (upper trace) and extracellular activity from the siphon nerve (lower trace) evoked by tactile stimulus (arrow). (B) Siphon tension (upper trace) and extracellular activity from the siphon nerve (lower trace) evoked by light stimulus (arrow). Scales: (A) 200 msec, 10 FV,tension 50 mV; (B) 1000 msec, 20 pV, tension 50 mV.
siphon nerve was cut ( N = 4 ) . I n other cases ( N = 11) the same procedure was followed except the response was habituated after the initial measurement and before the siphon nerve was cut. The mean response amplitude of the isolated preparation was 102% (S.D. = 11) of the response of the semi-isolated preparation. Thus, under these conditions there was no difference in response amplitude. The latency of the siphon contraction evoked by the light stimulus was much longer (generally 1100 msec) and more variable (500 to 2000 msec) than the latency t o the tactile stimulus. In individual preparations, removing the abdominal ganglion did not change the latency by more than 10%. Similarly the amplitude of the response evoked by the light stimulus ( n = 9) after removal of the abdominal ganglion was 99.5% (S.D. = 10) of the semi-intact preparation. Spontaneous contractions occurred in the semi-isolated and isolated preparations and were very similar to the contractions evoked by tactile or light stimulation except that in some preparations the spontaneous
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Fig. 3. Spontaneous siphon contractions in semi-isolated and isolated preparations. (A ) Semi-isolated preparation showing a spontaneous contraction preceded by a burst of activity in the siphon nerve and a second spontaneous contraction not preceded by a burst of activity. (B) Spontaneous contraction preceded by a burst of activity in the siphon nerve from a semi-isolated preparation. (C) The same preparation as B after the abdominal ganglion had been removed showing a spontaneous contraction without any preceding activity in the siphon nerve. Scales: 2 sec, 20 pV, and 100 mV for tension.
contractions were much larger than the evoked contractions. Some of the spontaneous contractions in the semi-isolated preparations were pl-eceded by a burst of activity in the siphon nerve, and some were not (Fig. 3). After the abdominal ganglion was removed these bursts of activity did not precede the spontaneous contractions (Fig. 3c), suggesting that the burst of activity was of central origin and initiated some of the spontaneous contractions but there is also a site of spontaneous activity in the periphery that produces the other spontaneous contractions. The frequency of spontaneous contractions was affected sometimes by removal o E the abdominaI ganglion but there was no clear trend. The involvement o F chemical synapses in these responses was tested by bathing the isolated preparations ( n = 4) in 0.1 m M calcium and 94 mM magnesium to block cliemical synaptic transmission in Aplysia (Halstead and Jacklet, 1974). The withdrawal response to tactile and light stimulation was reduced or abolished and the spontaneous contractions did not occur.
Habituation o f the siphon response The amplitude of the siphon withdrawal response decreased (habituated) with repeated light or tactile stimulation in the semi-isolated and isolated preparations. The normalized results obtained from 22 semiisolated and 29 isolated preparations are shown in Figure 4. There is no
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Fig. 4. Habituation of the siphon withdrawal response to repeated tactile stimulation in the semi-isolated and isolated preparations. (A) Pooled normalized data from 22 semi-isolated preparations to repeated tactile stimulation. (€3) Pooled normalized data from 29 isolated preparations to repeated tactile stimulation. The mean response and the standard deviation are shown.
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Fig. 5. Habituation and dishabituation of the siphon response. (A) Comparison of response decrement and dishabituation before and after removal (cut) of the abdominal ganglion and 120 min rest. Dishabituation (arrows) of the tactile response was brought about by the interposition of the light (L) stimulus. (B) Spontaneous recovery and habituation over series in a semi-isolated preparation to repeated tactile stimulation. (C) As in B, except in a n isolated preparation to light stimulation, dishabituation was by the interposition of the tactile ( H L O )stimulus (arrow).
apparent difference in either the rate or degree of response decrement exhibited by these preparations. This is also shown in a single preparation before and 120 min after removal of the abdominal ganglion in Figure 5A. Interposition of a novel stimulus in the stimulus series (light a t arrow, Fig. 5A) resulls in the immediate recovery or dishabituation of the response with or without the abdominal ganglion.
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Fig. 6. Spontaneous contractions and habituation in isolated preparations. (A) Changes in the amplitude of the siphon response to repeated tactile stimulation do not affect the amplitude of the spontaneous contractions. (B) The amplitude of the spontaneous contractions does not systematically change when they occur at a frequency of approximately 1 every 30 sec (the rate at which evoked responses habituate).
Spontaneous recovery of the response amplitude with rest occurs in both the semi-isolated (Fig. 5B) and the isolated (Fig. 5C) preparations. Longer rest times allow a greater recovery of the tactile response; as shown in Figure 5B, a 50 min rest produced 70% recovery and a 120 min rest produced 90% recovery. Similar results are shown in Figure 5C for recovery of the light response. More rapid habituation foIlowing spontaneous recovery (habituation over series) is also shown in the figures. The response of the siphon to either light or tactile stimulation in both the isolated and semi-isolated preparations exhibited similar rates of spontaneous recovery. Both the semi-isolated and isolated preparations also exhibited some of the other parametric characteristics of habituation. As can seen in Figure 5A and C the amount of recovery following successive presentations of the dishabitatory stimulus decreases. This phenomenon has been called habituation of dishabituation. Although not shown here below zero habituation and greater habituation with shorter ISI's are also exhibited by both preparations (Lukowiak, 1973). As mentioned above spontaneous contractions occur in both the semi-isolated and isolated preparations. These contractions continued during the habituation series but the amplitude of the spontaneous contractions was not affected by habituation of the evoked response in an isolated preparation and the spontaneous contractions had no apparent affect on the habituation of the evoked responses (Fig. 6A). Furthermore, the spontaneous contractions did not habituate when they occurred
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at frequencies (Fig. 6B) that would cause habituation of the evoked response. The events in Figure 6B represent one burst of spontaneous contractions that reoccurred a t intervals of approximately 10 min.
Responses of the siphon to electrical stimulation of the siphon nerve Electrical stimulation of the siphon nerve in both the semi-isolated and isolated preparations evoked activity in the nerve and resulted in a siphon withdrawal response (Fig. 7A). The response was similar to that which was evoked by light or tactile stimulation of the siphon. The amplitude
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Fig. 7. Siphon contraction evoked by electrical stimulation of the siphon nerve. (A) Siphon tension (upper trace) and extracellular activity recorded from the siphon nerve (lower trace) evoked by electrical stimulation of the siphon nerve in an isolated preparation. (B) Siphon tension evoked by electrical stimulation of the siphon nerve a t increasing stimulus intensity (20, 25, 30, 40, 50 volts) in a semi-isolated preparation. The same preparation as in B after the abdominal ganglion was removed. Scales: (A) 100 msec, 20 p v (B) and (C) 500 msec.
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and duration of the response was graded and depended on the stimulus intensity. The latency was usually between 200-300 msec and in any particular preparation did not change appreciably with changes in stimulus intensity. As shown in Figure 7B and C the stimulus latency, threshold, response amplitude and duration were similar before and after removal of the abdominal ganglion. However, a t higher stimulus intensities the semi-isolated preparation exhibited a secondary contraction (Figure 78, latency 1100 to 1500 msec). This secondary contraction was due to recurrent activation of central neurons since secondary contractions were never observed in isolated preparations.
Responses of the siphon with repeated electrical stimulation of the siphon nerve The amplitude of the siphon response to weak or moderate electrical stimulation of the siphon nerve decremented with repeated stimulation in both semi-isolated and isolated preparations. The pooled normalized data from 26 semi-isolated and 65 isolated preparations are shown in Figure 8A and B. There were no major differences in either the rate or degree of response decrement between preparations with and without the CNS. Data obtained from individual preparations before and after removal of the abdominal ganglion demonstrate also that both response decrement and recovery following the interposition of a novel stimulus (dishabituation) were similar (Fig. 9A). Some of the other characteristics of habituation (Thompson and Spencer, 1966) are shown in Figure 9B. Following response decrement with repeated stimulation the response recovered with rest, recovered to a greater extent with a longer rest, and the rate and degree of decrement were greater following the rest (habituation over series). The response exhibited enhancement followed by decrement if the stimulus was strong and the rate of decrement was faster for weaker stimuli. If the voltage was kept constant and the interstimulus interval was varied, the response decremented more rapidly with shorter intervals but at very short intervals (3 sec) the response showed enhancement followed by decrement. These data demonstrate that the repeated electrical stimulation of the siphon nerve in isolated A
B
Fig. 8. Changes in the amplitude of the siphon withdrawal response with repeated weak electrical stimulation of the siphon nerve in semi-isolated and isolated preparations. (A) Pooled normalized data (mean, f standard deviation) from 26 semi-isolated standard deviation) from 65 isopreparations. (B) Pooled normalized data (mean, lated preparations.
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Fig. 9. (A) Comparison of dishabituation in a preparation before and after removal of the abdominal ganglion. (A) The change in the amplitude of the siphon response to repeated electrical stimulation before and after removal of the abdominal ganglion and 80 min rest. Dishabituation (arrow) was brought about by the interposition of the tactile stimulus to the siphon. (B) Spontaneous recovery and habituation over series in a n isolated preparation.
and semi-isolated preparations results in habituation of the response and therefore there is a site of habituation at the peripheral terminals of the central axons.
Interaction between the peripheral and central neural circuits An interaction between central and peripheral neural circuits that mediate the siphon withdrawal response would be demonstrated if activity in one circuit affected the response mediated by the other circuit. Since electrical stimulation of the siphon nerve mimics the action of the central circuit in eliciting a siphon response, the dishabituation by a water drop of the response evoked by electrical stimulation of the siphon nerve (Fig. 9A) indicates a modulatory influence by the pkripheral system on the central system. Electrical stimulation of the siphon nerve dishabituates the response of the isolated siphon to water drops (Fig. 10A) and light (Fig. lOB), showing that the central system modulates the peripheral circuit also. The interaction between the circuits was always facilitatory. As shown in Figure lOC, electrical stimulation of the siphon nerve at a threshold level resulted in a slight response. Following the interposition of the tactile stimulus the response to the electrical stimulus was greatly augmented. The response then decremented with further stimulation with a time-course-like habituation. A second interposition of the tactile stimulus also produced enhancement, although it was not as great as that
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Fig. 10. Effects of activity in one circuit on the response mediated by the other circuit. (A) The change in amplitude of the siphon response in a n isolated preparation with repeated tactile stimulation Dishabituation (arrow) was brought about by the interposition of the electrical stimulus. (B) As in A, except the response was habituated with the light stimulus. ( C ) Enhancement of the siphon response to an initially subthreshold electrical stimulation by the interposition of the tactile stimulus. The response was also enhanced by the interposition of a stronger electrical stimulus (15 V, arrow) and by repeated electrical stimulation a t a higher frequency (2/sec for 2 sec signified by 4 X a t arrow).
following the first tactile stimulus. The response was also augmented by the interposition of a stronger electrical stimulus (15 V) or a brief increase in stimulus frequency (2,/sec for 2 sec). However, the greatest enhancement was produced by the tactile stimulus. Another approach was taken to demonstrate the interaction between the circuits. Tactile stimuli were repeatedly presented to the siphon of a semi-isolated preparation to habituate the response (Fig. 11). A sucrose block was then selectively applied to the siphon nerve to block axonal conduction while the siphon continued to be stimulated. The sucrose block isolated the central neurons from the effects of continued peripheral stimulation. When the block was released the siphon response was enhanced to 200% of the initial response amplitude. The response then decremented to a low level with repeated stimulation. This result demonstrates that the recovered central circuit has a facilitatory influence on the siphon response and is similar to dishabituation of the response by the interposition of a n electrical stimulus to the nerve. Responses of the isolated siphon evoked by each of the three stimulus types, tactile and light to the siphon and electrical stimulation of the siphon nerve were influenced by interposing either of the other two stimuli in a stimulus series. I n Figure 12, the second response to electrical stimulation of the siphon nerve (E) was enhanced following the interposition of the tactile stimulus (T). Similar experiments were performed using
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Fig. 11. Sucrose block experiment. The siphon response in a semi-isolated preparation was habituated with repeated tactile stimulation. Axonal conduction between the periphery and the abdominal ganglion was blocked by a sucrose flow while peripheral stimulation was continued. When the block was removed the response to tactile stimulation exhibited immediate recovery.
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Fig. 12. Enhancement of a n unhabituated response in an isolated preparation. The test stimulus (electrical stimulus, E) was presented, followed by the novel stimulus (tactile stimulus, T), and then the test stimulus again. The response t o the second test stimulus was enhanced by the novel stimulus. Time scale: 15 sec; tension 50 mV (60 mV = 1 8 ) .
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Fig. 13. Enhancement of unhabituated responses in isolated preparations-pooled data. Experiments performed similar t o t h a t in Fig. 12. Initial response to the test stimulus was taken as 100% and the response to the test stimulus following the novel stimulus as a percent of that. (L) light stimulus, (T) tactile stimulus, (E) electrical stimulus. IS1 = 35 sec.
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all possible stimulus combinations (electrical, light, and tactile) and all gave similar results (Fig. 13). I n all cases the unhabituated response was enhanced. These data demonstrate a facilitatory interaction between the central and peripheral circuits and also a facilitatory interaction between the neural circuits in the periphery that mediate the light and tactile response. The control experiments, repeated presentation of the same stimulus, did not show an enhancement of the response; rather the response decrement (Lukowiak, 1973). DISCUSSION
These results demonstrate that the abdominal ganglion (central neurons), which is connected to the siphon by the siphon nerve, contributes very little to the siphon response or habituation of the response under the stimulus conditions of these experiments. Response amplitudes and latencies to water drops and light stimuli were virtually unchanged following removal of the abdominal ganglion. Habituation in the isolated siphon has all the characteristics that have been reported for habituation in intact and semi-intact Aplysia (Kupfermann e t al., 1970; Pinsker e t al., 1970; Carew, Castellucci and Kandel, 1972). This includes all three important ones (exponential decline in response, spontaneous recovery and dishabituation) as well as most of the other characteristics outlined by Thompson and Spencer (1966) for vertebrate habituation. Therefore, the peripheral neural circuit of the siphon is competent to mediate habituation and dishabituation independent of the central neurons when the stimulus is weak. I n a related mollusk, Spisula, weak tactile stimulation of the siphon results in a local contraction and stronger stimulation resulted in siphon withdrawal and eventual shell closure. Removal of the visceral ganglion abolished the withdrawal but did not affect the local contractions which were mediated by peripheral neurons, which show antifacilitation (Prior, 1972a,b). Siphon withdrawal (Kupfermann and Kandel, 1969) and habituation of this response (Carew et al., 1972) have been studied in intact and semiintact animals. These investigators claimed that the responses were mediated by central neurons. However, they used a strong tactile stimulus (jet of seawater) compared t o the weaker (water drop) used in our experiments. The stronger stimulus activates neurons in the abdominal ganglion that produce a secondary response component in addition to that mediated by the peripheral neurons. Such secondary responses were evoked when the siphon nerve was stimulated electrically in semi-isolated preparations. The response evoked by electrical stimulation was similar to the response evoked by tactile or light stimulation of the siphon directly. The electrically evoked contraction was graded in amplitude according to the applied voltage but the latency was constant. With the abdominal
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ganglion attached the response amplitude was not different than the amplitude without the ganglion, except for the secondary contractions in the semi-isolated preparations evoked at higher stimulus intensikies. Electrical stimulation of the whole siphon nerve activates the efferent axons (motor) as well as afferent axons (sensory) to the abdominal ganglion. If the peripheral and central circuits are parallel and independent, one would expect that there would be no interaction between the two systems. Our results show that there is extensive interaction that appears to be exclusively excitatory. The response decrement with repeated electrical stimulation of the siphon nerve in the isolated preparation is an example of habituation since most of the parametric characteristics of habituation (Thompson and Spencer, 1966) have been demonstrated. Thus, the efferent fibers from the central neurons have a locus of habituation in the periphery. This site of habituation could be either at the neuromuscular junction (Bruner and Kennedy, 1970), if the efferents in the siphon nerve are indeed motor axons, or at the central-peripheral neural plexus synapse. Since the decremented response with repeated electrical stimulation is dishabituated by tactile or light stimulation and since dishabituation over a separate pathway is generally a property of neuronal synpases and not neuromuscular junctions, it appears likely that the central efferents act via a peripheral neural plexus. At this site strong stimulation and stimulation a t intervals less than 5 sec produce response sensitization. These data are in agreement with the notion of Groves and Thompson (1970) that habituation is the reflection of opposing processes, decremental and incremental. Varying the stimulus parameters can allow one process to dominate the other and result in response decrement or increment. The nature of dishabituation in the isolated siphon is a facilitatory process and not the removal of a habituation process. This is evident from some of the results presented above, where electrical stimulation a t a threshold level produced no response but following the interposition of a tactile stimulus a response was elicited. Since no response was evoked initially the tactile stimulus must act to facilitate the circuit. The interposition of the tactile stimulus was operationally similar to the presentation of the dishabituatory stimulus. The independence of habituation and dishabituation processes is in agreement with the earlier conclusions of Thompson and Spencer (1966) in the cat spinal cord and Carew e t al. (1971) in the CNS of Aplysia. Our data also demonstrate that the peripheral neural circuit is competent to mediate response sensitization in addition to sensitization mediated by central neurons (Pinsker, Hening, Carew and Kandel, 1973). Sucrose block and other types of reversible block experiments have been used by other investigators (Bruner and Kehoe, 1970; Castellucci et al., 1970) to determine if the site of habituation was central or peripheral.
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Although their results were similar to those obtained above for the siphon response, they interpreted their results to indicate that habituation was a central phenomenon. However, they did not test for peripheral habituation as we have demonstrated in the siphon. Our findings suggest that the recovered central circuit has a facilitatory effect on the habituated peripheral neural circuit similar to the interposition of a novel stimulus. The question of a n interaction between central and peripheral neural circuits in the mediation of habituation in Aplysia has been addressed. Kupfermann, Pinsker, Castellucci and Kandel (1971) found that there was no transfer or generalization of habituation between the central and peripheral neural circuits that mediate gill withdrawal and concluded that the central and peripheral neural circuits are parallel and independent. However, nongeneralization of habituation occurs within the central neural circuit when two different afferent pathways are stimulated (Carew et al., 1971). Thus one might expect nongeneralization t o occur between the central and peripheral neural circuits since the afferent pathways are different. Peretz (1970) demonstrated that electrical stimulation of the ctendial nerve dishabituates the gill withdrawal response to tactile stimulation of a gill pinnule in a n isolated preparation. Recently, Lukowiak (unpublished observation) observed that tactile stimulation of the siphon dishabituates the gill response that had been habituated with repeated tactile stimulation of the pinnule. Thus activity in the central neural circuit conducted to the gill over the ctendial nerve appears to have a facilitatory influence on the peripheral neural circuit in the gill. An inhibitory influence of the central circuit on the peripheral neural circuit in the gill has also been demonstrated (Peretz and Howieson, 1973). Further details on the involvement of central and peripheral circuits in gill and siphon habituation may be found in a recent review (Jacklet and Lukowiak, 1974). Our results demonstrate that each neural circuit that mediates siphon behavior can modulate the response mediated by the other neural circuit. Thus, a stimulus to the siphon of the intact animal may evoke a response directly by the peripheral circuit but, additionally, information is sent to the central nervous system which may mediate a n additional withdrawal depending upon the state of the system. Also, responses mediated by central neurons may be modulated by the peripheral circuit depending upon its state. There are three sites of habituation, one in periphery between receptor and effector, one in the central reflex pathway and one at the peripheral terminals of the central projections. The central circuit may be responsible for long-term retention of habituation (Carew, Pinsker and Kandel, 1972) although it has been found that there is no difference in retention of short-term habituation between preparations with or without the central neurons (Lukowiak, 1973). Spontaneous contractions of the siphon and gill have been previously reported for intact and semi-intact preparations and attributed to the
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activity of neurons in the abdominal ganglion (Kupfermann and Kandel, 1969; Peretz, 1969). We have also seen abdominal ganglion neuron activity correlated with spontaneous contractions (Lukowiak, 1973) and a burst of activity in the siphon nerve preceding spontaneous contractions. Additionally, spontaneous contractions occur in the isolated siphon. Spontaneous contractions do not influence habituation of evoked responses in the isolated siphon or reflex contractions of the gill (Pinsker et al., 1970) but they may infrequently dishabituate gill pinnule responses (Peretz, 1970). Histological examination of the branch points of the siphon nerve in the siphon shows that there are many neurons, 10-50 p in diameter, in the nerve (Jacklet and Lukowiak, 1974). Also, these neurons have been selectively back-filled with cobalt through peripheral branches of the siphon nerve. These neurons along with other less clearly defined nerve net elements must mediate the siphon responses. Neurons distributed along peripheral nerve trunks are common in mollusks (Gorman and Mirolli, 1969; Prior, 1972a,b; Peretz and Estes, 1974), and a peripheral nerve net mediates responses (Hoyle and Willows, 1973). We propose a scheme for the peripheral neural circuit in the siphon that includes two receptors (tactile and photic) because the response latencies to tactile and light stimuli differ by a factor of 10 and tactile stimuli evoke an obvious burst of activity in the siphon nerve and light does not. There is evidence for mechanoreceptors and photoreceptors on the siphon of gastropods (Dijkgraaf, 1935; Bailey and Laverack, 1966; Millott, 1968; Crisp, 1972; Newby, 1973). The receptors synapse on peripheral neural elements and, additionally, send tactile information directly to the CNS. The peripheral neural plexus (PNP) must include motor neurons as well. Evidence for chemical synapses in the P N P is reported by Newby (1973) in the sensitivity of the light response to many presumed transmitter substances and the block of responses by high magnesium. The actual mechanisms of habituation and dishabituation are unknown but they may include chemical synaptic mechanisms such as homosynaptic depression and heterosynaptic facilitation (Kandel and Spencer, 1968; Kandel, Castellucci, Pinsker and Kupfermann, 1970; Carew et al., 1971; Kandel, 1974) that could operate at the receptor-neuron synapse or the neuromuscular synapse. The central neurons may terminate on the P N P and siphon musculature since this is shown to be a site of habituation and dishabituation. Adaptive siphon behavior is influenced by the state of the peripheral and central neural circuits, so any complete analysis of the neural correlates of this response must take into account the peripheral as well as the central sites of plasticity. This research was supported by National Institutes of Health Grant NS 08443 to
J.W.J.
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REFERENCES
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LUKOWIA K AND JACKLE T KUPFERMANN, I., CASTELLUCCI, V., PINSKER,H., and KANDEL, E. R. (1970). Neurorlal correlates of habituation and dishabituation of the gill withdrawal reflex in Aplysia. Science 167: 1743-1745. I., PINSKER,H., CASTELLUCCI, V., and KANDEL,E. R. (1971) Central KUPFERMANN, and peripheral control of gill movements in Aplysia. Science 174: 1252-1256. LUKOWIAK, K. (1973). Peripheral and central neural processes in habituation of reflexes in Aplysia. Ph.D. Thesis, S.U.N.Y. Albany. LUKOWIAK, K. and JACKLET, J. W. (1972). Habituation and dishabituation: Interactions between peripheral and central nervous systems in Aplysia. Science 178: 1306-1308. MILLOTT,N. (1968). The dermal light sense. Symp. Zool. Soc. Lond. 23: 1-36. NEWRY,N. A. (1973). Habituation to light and spontaneous activity in the isolated siphon of Aplysia: Pharmacological observations. Compt. Gen. Pharmacol. 4: 9 1100. PERETZ,B. (1969). Central neuron initiation of periodic gill movements. Science 166: 1167-1172. PERETZ,B. (1970). Habituation and dishabituation in the absence of a central nervous system. Science 169: 379-381. PERETZ, B. and ESTES, J. (1974). Histology and histochemistry of the peripheral neural plexus in the Aplysia gill. J . Neurobiol. 5(1): 3-19. PERETZ,B. and HOWIESON, D. B. (1973). Central influence on peripherally mediated habituation of an Aplysia gill withdrawal response. J. Comp. Physiol. 84: 1-18. PERETZ, B. and MOLLER,R. A. (1972). Regulation of habituated withdrawal response by a ganglion in the Aplysia gill. Amer. Zool. 12: 693. I., CASTELLUCCI, V., and KANDEL, E. R. (1970). HabituaPINSKER,H., KUPFERMANN, tion and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167: 17401742. PINSKER, H., HENING,W. A., CAREW,T. J., and KANDEL,E. R. (1973). Long-term sensitization of a defensive withdrawal reflex in Aplysia. Science 182: 1039-1042. PRIOR,D. J. (1972a). Electrophysiological analysis of peripheral neurons and their possible role in the local reflexes of a mollusc. J . Exp. Biol. 57: 133-145. PRIOR,D. J. (197213). A neural correlate of behavioral stimulus intensity discrimination in a mollusc. J . Exp. Biol. 57: 147-160. PROSSER,C. L. and HUNTER,W. S. (1936). The extinction of startle responses and spinal reflexes in the white rat. Amer. J . Physiol. 117: 609-618. R. F. and SPENCER, W. A. (1966) Habituation: A model phenomenon for THOMPSON, the study of neuronal substrates of behavior. Psychol. Reu. 73: 16-43. THOMPSON, R. F., PATTERSON, M. M., and TEYLER,T. J. (1972). The neurophysiology of learning. Annu. Reu. Psychol. 23: 73-104.
Accepted for publication August 1, 1974
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HABITUATION AND D ISHAB ITUATI ON MEDIATED BY THE PERIPHERAL AND CENTRAL NEURAL CIRCUITS O F THE SIPHON OF APLYSIA K E N N E T H LUKOWIAK and JON JACKLET Department of Biological Sciences and Neurobiology Research Center, State University of New York at Albany, Albany, New York 12222
SUMMARY
The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Responses evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems. INTRODUCTION
Habituation, perhaps the most elementary and ubiquitous form of behavioral plasticity, has been defined as a decrease in response to repeated stimulation (Harris, 1943). More explicitly, habituation in invertebrate preparations has been operationally defined by a set of 9 parametric characteristics (Thompson and Spencer, 1966). The three characteristics that are generally sufficient to establish that a given response habituates are: 1)exponential decrease in response strength to repeated stimulation, 2) spontaneous recovery, and 3 ) dishabituation (Eisenstein and Peretz, 1973). Habituation of spinal reflexes has been shown to occur in the absence of higher neural centers (Prosser and Hunter, 1936) in polysynaptic pathways of the spinal cord (reviewed by Thompson and Spencer, 1966; Griffin, 1970; Thompson, Patterson and Teyler, 1972). In the invertebrate Aplysia, habituation of siphon and gill withdrawal has been at183 @ 1975 by John Wiley & Sons, Inc.
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tributed to changes in the activity of central neurons (Castellucci, Pinsker, Kupfermann and Kandel, 1970; Kupfermann, Castellucci, Pinsker and Kandel, 1970; Pinsker, Kupfermann, Castellucci and Kandel, 1970). However, the responses (gill and siphon withdrawal) that were examined have been shown to undergo habituation in the absence of central neurons (Peretz, 1970, Lukowiak and Jacklet, 1972). These results raise the question of how much each system, central and peripheral, contributes to the withdrawal responses. We examined the characteristics of habituation and dishabituation in the isolated siphon, the contribution of the central neurons to the siphon response, and the modulatory influences between the peripheral and central circuits. METHODS
Sixty Aplysia californica, obtained from Pacific-Bio-Marine (Venice, Calif.) were used in these experiments. The animals were maintained prior to use in artificial seawater (Instant Ocean) a t 15-17°C under a light-dark cycle of 13 hr light and 11 hr dark. I n preparation the animal was pinned out dorsal side up on a tray and a n incision was made just rostra1 to the mantle. T h e pleural-abdominal connectives were cut and the siphon, mantle, gill, and abdominal ganglion complex were cut free from the animal. This complex was reduced to the two preparations (Fig. I) used in this study: (I) the siphon-mantle connected to the abdominal ganglion by the siphon nerve only (the semiisolated preparation) and (2) the siphon without the abdominal ganglion (the isolated preparation). The preparations were pinned out completely submerged under artificial seawater (19-22°C) in a chamber with a Sylgard 184 (Dow-Corning, Midland, Mich.) Retractor Abdominal Ganglion
Fig. 1. Diagrammatic representation of the semi-isolated preparation. The abdominal ganglion is attached to the siphon, mantle, and gill by the siphon nerve. EXtracellular recordings were made from the siphon nerve and intracellular recordings were made from neurons in the abdominal ganglion. The siphon nerve was stimulated electrically (n).The nerve was severed at the arrow to make a n isolated preparation. S.N., siphon nerve; L.C. and R.C., left and right connectives; B.N., branchial nerve.
(4)
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base. A thread was attached from the siphon to a Grass Tension Transducer (Ft 03) and withdrawal was measured as a voltage deflection (60 mV = 1 g) on either a polygraph or a storage oscilloscope. Neuronal activity in the siphon nerve was monitored by extracellular recordings with suction electrodes (made en passant in the semi-isolated preparations) or electrical stimulation with 1 msec square pulses was delivered through the same electrodes. Light and tactile stimuli were used to evoke the withdrawal response of the siphon. The light stimulus was approximately 1000 lumen,/mZand 6 sec in duration from a n incandescent lamp. The tactile stimulus was a single drop of seawater falling from a height of 5 cm. The stimuli were presented with an interstimulus interval (ISI), usually of 30 sec. The preparations were set up in the chamber and then allowed to rest for a t least 45 min before testing was begun. I n some experiments isotonic sucrose (0.8 M ) was used to block axonal conduction in the siphon nerve in order to isolate the central neurons in the abdominal ganglion from the effects of peripheral stimulation. Isotonic sucrose solution was flowed over the siphon nerve from polyethylene tubing a t a slow rate and was drawn off by another tube. The sucrose displaced the ions necessary for the propagation of the action potential and caused a block that was confirmed by extracellular recordings from the siphon nerve. The block was relieved within 30 sec after cessation of the sucrose flow. RESULTS
Siphon withdrawal responses The semi-isolated and isolated siphon preparations respond to tactile or light stimulation. The response consisted of longitudinal contraction of the siphon, so that the tip of the siphon is withdrawn toward the mantle, and a concomitant constriction of the siphon lumen. I n these experiments we measured the tension developed by longitudinal contraction. The tension evoked by a tactile stimulus in an isolated preparation is shown in Figure 2A. The stimultaneous extracellular recording from the siphon nerve indicates that afferent activity was evoked by the stimulus and occurred with a latency similar to that for tension. This afferent activity would presumably activate centra! neurons. Figure 2 B shows the response evoked by light stimulation of an isolated preparation. Note that the extracellular recording from the siphon nerve revealed very little activity associated with the light stimulus. This has been a consistent finding. The duration and amplitude of the responses depends upon the particular preparation and the history of stimulation; however, in the rested condition the tension was about 3-5 g for light or tactile stimulation. A comparison was made between the latency of the siphon contraction to the tactile stimulus in the semi-isolated and the isolated preparations. The mean latency in the isolated preparations ( n = 36, 18 preparations) was 118 msec (S.D. = 5.9) while the mean latency in the semi-isolated preparations ( n = 40, 32 preparations) was 119 msec (S.D. = 4.2). Thus, there is no difference in the latency. Also the response amplitudes evoked by tactile stimulation were compared in the same preparation with and without the abdominal ganglion. The response of the semiisolated preparation was measured after a 1hr rest and again 1hr after the
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Fig. 2. Comparison of the latency of the siphon contraction and evoked neural activity in isolated preparations to tactile and light stimulation. (A) Siphon tension (upper trace) and extracellular activity from the siphon nerve (lower trace) evoked by tactile stimulus (arrow). (B) Siphon tension (upper trace) and extracellular activity from the siphon nerve (lower trace) evoked by light stimulus (arrow). Scales: (A) 200 msec, 10 FV,tension 50 mV; (B) 1000 msec, 20 pV, tension 50 mV.
siphon nerve was cut ( N = 4 ) . I n other cases ( N = 11) the same procedure was followed except the response was habituated after the initial measurement and before the siphon nerve was cut. The mean response amplitude of the isolated preparation was 102% (S.D. = 11) of the response of the semi-isolated preparation. Thus, under these conditions there was no difference in response amplitude. The latency of the siphon contraction evoked by the light stimulus was much longer (generally 1100 msec) and more variable (500 to 2000 msec) than the latency t o the tactile stimulus. In individual preparations, removing the abdominal ganglion did not change the latency by more than 10%. Similarly the amplitude of the response evoked by the light stimulus ( n = 9) after removal of the abdominal ganglion was 99.5% (S.D. = 10) of the semi-intact preparation. Spontaneous contractions occurred in the semi-isolated and isolated preparations and were very similar to the contractions evoked by tactile or light stimulation except that in some preparations the spontaneous
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Fig. 3. Spontaneous siphon contractions in semi-isolated and isolated preparations. (A ) Semi-isolated preparation showing a spontaneous contraction preceded by a burst of activity in the siphon nerve and a second spontaneous contraction not preceded by a burst of activity. (B) Spontaneous contraction preceded by a burst of activity in the siphon nerve from a semi-isolated preparation. (C) The same preparation as B after the abdominal ganglion had been removed showing a spontaneous contraction without any preceding activity in the siphon nerve. Scales: 2 sec, 20 pV, and 100 mV for tension.
contractions were much larger than the evoked contractions. Some of the spontaneous contractions in the semi-isolated preparations were pl-eceded by a burst of activity in the siphon nerve, and some were not (Fig. 3). After the abdominal ganglion was removed these bursts of activity did not precede the spontaneous contractions (Fig. 3c), suggesting that the burst of activity was of central origin and initiated some of the spontaneous contractions but there is also a site of spontaneous activity in the periphery that produces the other spontaneous contractions. The frequency of spontaneous contractions was affected sometimes by removal o E the abdominaI ganglion but there was no clear trend. The involvement o F chemical synapses in these responses was tested by bathing the isolated preparations ( n = 4) in 0.1 m M calcium and 94 mM magnesium to block cliemical synaptic transmission in Aplysia (Halstead and Jacklet, 1974). The withdrawal response to tactile and light stimulation was reduced or abolished and the spontaneous contractions did not occur.
Habituation o f the siphon response The amplitude of the siphon withdrawal response decreased (habituated) with repeated light or tactile stimulation in the semi-isolated and isolated preparations. The normalized results obtained from 22 semiisolated and 29 isolated preparations are shown in Figure 4. There is no
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Fig. 4. Habituation of the siphon withdrawal response to repeated tactile stimulation in the semi-isolated and isolated preparations. (A) Pooled normalized data from 22 semi-isolated preparations to repeated tactile stimulation. (€3) Pooled normalized data from 29 isolated preparations to repeated tactile stimulation. The mean response and the standard deviation are shown.
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Fig. 5. Habituation and dishabituation of the siphon response. (A) Comparison of response decrement and dishabituation before and after removal (cut) of the abdominal ganglion and 120 min rest. Dishabituation (arrows) of the tactile response was brought about by the interposition of the light (L) stimulus. (B) Spontaneous recovery and habituation over series in a semi-isolated preparation to repeated tactile stimulation. (C) As in B, except in a n isolated preparation to light stimulation, dishabituation was by the interposition of the tactile ( H L O )stimulus (arrow).
apparent difference in either the rate or degree of response decrement exhibited by these preparations. This is also shown in a single preparation before and 120 min after removal of the abdominal ganglion in Figure 5A. Interposition of a novel stimulus in the stimulus series (light a t arrow, Fig. 5A) resulls in the immediate recovery or dishabituation of the response with or without the abdominal ganglion.
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s SPONTANEOUS
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Fig. 6. Spontaneous contractions and habituation in isolated preparations. (A) Changes in the amplitude of the siphon response to repeated tactile stimulation do not affect the amplitude of the spontaneous contractions. (B) The amplitude of the spontaneous contractions does not systematically change when they occur at a frequency of approximately 1 every 30 sec (the rate at which evoked responses habituate).
Spontaneous recovery of the response amplitude with rest occurs in both the semi-isolated (Fig. 5B) and the isolated (Fig. 5C) preparations. Longer rest times allow a greater recovery of the tactile response; as shown in Figure 5B, a 50 min rest produced 70% recovery and a 120 min rest produced 90% recovery. Similar results are shown in Figure 5C for recovery of the light response. More rapid habituation foIlowing spontaneous recovery (habituation over series) is also shown in the figures. The response of the siphon to either light or tactile stimulation in both the isolated and semi-isolated preparations exhibited similar rates of spontaneous recovery. Both the semi-isolated and isolated preparations also exhibited some of the other parametric characteristics of habituation. As can seen in Figure 5A and C the amount of recovery following successive presentations of the dishabitatory stimulus decreases. This phenomenon has been called habituation of dishabituation. Although not shown here below zero habituation and greater habituation with shorter ISI's are also exhibited by both preparations (Lukowiak, 1973). As mentioned above spontaneous contractions occur in both the semi-isolated and isolated preparations. These contractions continued during the habituation series but the amplitude of the spontaneous contractions was not affected by habituation of the evoked response in an isolated preparation and the spontaneous contractions had no apparent affect on the habituation of the evoked responses (Fig. 6A). Furthermore, the spontaneous contractions did not habituate when they occurred
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at frequencies (Fig. 6B) that would cause habituation of the evoked response. The events in Figure 6B represent one burst of spontaneous contractions that reoccurred a t intervals of approximately 10 min.
Responses of the siphon to electrical stimulation of the siphon nerve Electrical stimulation of the siphon nerve in both the semi-isolated and isolated preparations evoked activity in the nerve and resulted in a siphon withdrawal response (Fig. 7A). The response was similar to that which was evoked by light or tactile stimulation of the siphon. The amplitude
A
B
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Fig. 7. Siphon contraction evoked by electrical stimulation of the siphon nerve. (A) Siphon tension (upper trace) and extracellular activity recorded from the siphon nerve (lower trace) evoked by electrical stimulation of the siphon nerve in an isolated preparation. (B) Siphon tension evoked by electrical stimulation of the siphon nerve a t increasing stimulus intensity (20, 25, 30, 40, 50 volts) in a semi-isolated preparation. The same preparation as in B after the abdominal ganglion was removed. Scales: (A) 100 msec, 20 p v (B) and (C) 500 msec.
(c)
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and duration of the response was graded and depended on the stimulus intensity. The latency was usually between 200-300 msec and in any particular preparation did not change appreciably with changes in stimulus intensity. As shown in Figure 7B and C the stimulus latency, threshold, response amplitude and duration were similar before and after removal of the abdominal ganglion. However, a t higher stimulus intensities the semi-isolated preparation exhibited a secondary contraction (Figure 78, latency 1100 to 1500 msec). This secondary contraction was due to recurrent activation of central neurons since secondary contractions were never observed in isolated preparations.
Responses of the siphon with repeated electrical stimulation of the siphon nerve The amplitude of the siphon response to weak or moderate electrical stimulation of the siphon nerve decremented with repeated stimulation in both semi-isolated and isolated preparations. The pooled normalized data from 26 semi-isolated and 65 isolated preparations are shown in Figure 8A and B. There were no major differences in either the rate or degree of response decrement between preparations with and without the CNS. Data obtained from individual preparations before and after removal of the abdominal ganglion demonstrate also that both response decrement and recovery following the interposition of a novel stimulus (dishabituation) were similar (Fig. 9A). Some of the other characteristics of habituation (Thompson and Spencer, 1966) are shown in Figure 9B. Following response decrement with repeated stimulation the response recovered with rest, recovered to a greater extent with a longer rest, and the rate and degree of decrement were greater following the rest (habituation over series). The response exhibited enhancement followed by decrement if the stimulus was strong and the rate of decrement was faster for weaker stimuli. If the voltage was kept constant and the interstimulus interval was varied, the response decremented more rapidly with shorter intervals but at very short intervals (3 sec) the response showed enhancement followed by decrement. These data demonstrate that the repeated electrical stimulation of the siphon nerve in isolated A
B
Fig. 8. Changes in the amplitude of the siphon withdrawal response with repeated weak electrical stimulation of the siphon nerve in semi-isolated and isolated preparations. (A) Pooled normalized data (mean, f standard deviation) from 26 semi-isolated standard deviation) from 65 isopreparations. (B) Pooled normalized data (mean, lated preparations.
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Fig. 9. (A) Comparison of dishabituation in a preparation before and after removal of the abdominal ganglion. (A) The change in the amplitude of the siphon response to repeated electrical stimulation before and after removal of the abdominal ganglion and 80 min rest. Dishabituation (arrow) was brought about by the interposition of the tactile stimulus to the siphon. (B) Spontaneous recovery and habituation over series in a n isolated preparation.
and semi-isolated preparations results in habituation of the response and therefore there is a site of habituation at the peripheral terminals of the central axons.
Interaction between the peripheral and central neural circuits An interaction between central and peripheral neural circuits that mediate the siphon withdrawal response would be demonstrated if activity in one circuit affected the response mediated by the other circuit. Since electrical stimulation of the siphon nerve mimics the action of the central circuit in eliciting a siphon response, the dishabituation by a water drop of the response evoked by electrical stimulation of the siphon nerve (Fig. 9A) indicates a modulatory influence by the pkripheral system on the central system. Electrical stimulation of the siphon nerve dishabituates the response of the isolated siphon to water drops (Fig. 10A) and light (Fig. lOB), showing that the central system modulates the peripheral circuit also. The interaction between the circuits was always facilitatory. As shown in Figure lOC, electrical stimulation of the siphon nerve at a threshold level resulted in a slight response. Following the interposition of the tactile stimulus the response to the electrical stimulus was greatly augmented. The response then decremented with further stimulation with a time-course-like habituation. A second interposition of the tactile stimulus also produced enhancement, although it was not as great as that
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Fig. 10. Effects of activity in one circuit on the response mediated by the other circuit. (A) The change in amplitude of the siphon response in a n isolated preparation with repeated tactile stimulation Dishabituation (arrow) was brought about by the interposition of the electrical stimulus. (B) As in A, except the response was habituated with the light stimulus. ( C ) Enhancement of the siphon response to an initially subthreshold electrical stimulation by the interposition of the tactile stimulus. The response was also enhanced by the interposition of a stronger electrical stimulus (15 V, arrow) and by repeated electrical stimulation a t a higher frequency (2/sec for 2 sec signified by 4 X a t arrow).
following the first tactile stimulus. The response was also augmented by the interposition of a stronger electrical stimulus (15 V) or a brief increase in stimulus frequency (2,/sec for 2 sec). However, the greatest enhancement was produced by the tactile stimulus. Another approach was taken to demonstrate the interaction between the circuits. Tactile stimuli were repeatedly presented to the siphon of a semi-isolated preparation to habituate the response (Fig. 11). A sucrose block was then selectively applied to the siphon nerve to block axonal conduction while the siphon continued to be stimulated. The sucrose block isolated the central neurons from the effects of continued peripheral stimulation. When the block was released the siphon response was enhanced to 200% of the initial response amplitude. The response then decremented to a low level with repeated stimulation. This result demonstrates that the recovered central circuit has a facilitatory influence on the siphon response and is similar to dishabituation of the response by the interposition of a n electrical stimulus to the nerve. Responses of the isolated siphon evoked by each of the three stimulus types, tactile and light to the siphon and electrical stimulation of the siphon nerve were influenced by interposing either of the other two stimuli in a stimulus series. I n Figure 12, the second response to electrical stimulation of the siphon nerve (E) was enhanced following the interposition of the tactile stimulus (T). Similar experiments were performed using
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Fig. 11. Sucrose block experiment. The siphon response in a semi-isolated preparation was habituated with repeated tactile stimulation. Axonal conduction between the periphery and the abdominal ganglion was blocked by a sucrose flow while peripheral stimulation was continued. When the block was removed the response to tactile stimulation exhibited immediate recovery.
E
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Fig. 12. Enhancement of a n unhabituated response in an isolated preparation. The test stimulus (electrical stimulus, E) was presented, followed by the novel stimulus (tactile stimulus, T), and then the test stimulus again. The response t o the second test stimulus was enhanced by the novel stimulus. Time scale: 15 sec; tension 50 mV (60 mV = 1 8 ) .
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Fig. 13. Enhancement of unhabituated responses in isolated preparations-pooled data. Experiments performed similar t o t h a t in Fig. 12. Initial response to the test stimulus was taken as 100% and the response to the test stimulus following the novel stimulus as a percent of that. (L) light stimulus, (T) tactile stimulus, (E) electrical stimulus. IS1 = 35 sec.
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all possible stimulus combinations (electrical, light, and tactile) and all gave similar results (Fig. 13). I n all cases the unhabituated response was enhanced. These data demonstrate a facilitatory interaction between the central and peripheral circuits and also a facilitatory interaction between the neural circuits in the periphery that mediate the light and tactile response. The control experiments, repeated presentation of the same stimulus, did not show an enhancement of the response; rather the response decrement (Lukowiak, 1973). DISCUSSION
These results demonstrate that the abdominal ganglion (central neurons), which is connected to the siphon by the siphon nerve, contributes very little to the siphon response or habituation of the response under the stimulus conditions of these experiments. Response amplitudes and latencies to water drops and light stimuli were virtually unchanged following removal of the abdominal ganglion. Habituation in the isolated siphon has all the characteristics that have been reported for habituation in intact and semi-intact Aplysia (Kupfermann e t al., 1970; Pinsker e t al., 1970; Carew, Castellucci and Kandel, 1972). This includes all three important ones (exponential decline in response, spontaneous recovery and dishabituation) as well as most of the other characteristics outlined by Thompson and Spencer (1966) for vertebrate habituation. Therefore, the peripheral neural circuit of the siphon is competent to mediate habituation and dishabituation independent of the central neurons when the stimulus is weak. I n a related mollusk, Spisula, weak tactile stimulation of the siphon results in a local contraction and stronger stimulation resulted in siphon withdrawal and eventual shell closure. Removal of the visceral ganglion abolished the withdrawal but did not affect the local contractions which were mediated by peripheral neurons, which show antifacilitation (Prior, 1972a,b). Siphon withdrawal (Kupfermann and Kandel, 1969) and habituation of this response (Carew et al., 1972) have been studied in intact and semiintact animals. These investigators claimed that the responses were mediated by central neurons. However, they used a strong tactile stimulus (jet of seawater) compared t o the weaker (water drop) used in our experiments. The stronger stimulus activates neurons in the abdominal ganglion that produce a secondary response component in addition to that mediated by the peripheral neurons. Such secondary responses were evoked when the siphon nerve was stimulated electrically in semi-isolated preparations. The response evoked by electrical stimulation was similar to the response evoked by tactile or light stimulation of the siphon directly. The electrically evoked contraction was graded in amplitude according to the applied voltage but the latency was constant. With the abdominal
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ganglion attached the response amplitude was not different than the amplitude without the ganglion, except for the secondary contractions in the semi-isolated preparations evoked at higher stimulus intensikies. Electrical stimulation of the whole siphon nerve activates the efferent axons (motor) as well as afferent axons (sensory) to the abdominal ganglion. If the peripheral and central circuits are parallel and independent, one would expect that there would be no interaction between the two systems. Our results show that there is extensive interaction that appears to be exclusively excitatory. The response decrement with repeated electrical stimulation of the siphon nerve in the isolated preparation is an example of habituation since most of the parametric characteristics of habituation (Thompson and Spencer, 1966) have been demonstrated. Thus, the efferent fibers from the central neurons have a locus of habituation in the periphery. This site of habituation could be either at the neuromuscular junction (Bruner and Kennedy, 1970), if the efferents in the siphon nerve are indeed motor axons, or at the central-peripheral neural plexus synapse. Since the decremented response with repeated electrical stimulation is dishabituated by tactile or light stimulation and since dishabituation over a separate pathway is generally a property of neuronal synpases and not neuromuscular junctions, it appears likely that the central efferents act via a peripheral neural plexus. At this site strong stimulation and stimulation a t intervals less than 5 sec produce response sensitization. These data are in agreement with the notion of Groves and Thompson (1970) that habituation is the reflection of opposing processes, decremental and incremental. Varying the stimulus parameters can allow one process to dominate the other and result in response decrement or increment. The nature of dishabituation in the isolated siphon is a facilitatory process and not the removal of a habituation process. This is evident from some of the results presented above, where electrical stimulation a t a threshold level produced no response but following the interposition of a tactile stimulus a response was elicited. Since no response was evoked initially the tactile stimulus must act to facilitate the circuit. The interposition of the tactile stimulus was operationally similar to the presentation of the dishabituatory stimulus. The independence of habituation and dishabituation processes is in agreement with the earlier conclusions of Thompson and Spencer (1966) in the cat spinal cord and Carew e t al. (1971) in the CNS of Aplysia. Our data also demonstrate that the peripheral neural circuit is competent to mediate response sensitization in addition to sensitization mediated by central neurons (Pinsker, Hening, Carew and Kandel, 1973). Sucrose block and other types of reversible block experiments have been used by other investigators (Bruner and Kehoe, 1970; Castellucci et al., 1970) to determine if the site of habituation was central or peripheral.
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Although their results were similar to those obtained above for the siphon response, they interpreted their results to indicate that habituation was a central phenomenon. However, they did not test for peripheral habituation as we have demonstrated in the siphon. Our findings suggest that the recovered central circuit has a facilitatory effect on the habituated peripheral neural circuit similar to the interposition of a novel stimulus. The question of a n interaction between central and peripheral neural circuits in the mediation of habituation in Aplysia has been addressed. Kupfermann, Pinsker, Castellucci and Kandel (1971) found that there was no transfer or generalization of habituation between the central and peripheral neural circuits that mediate gill withdrawal and concluded that the central and peripheral neural circuits are parallel and independent. However, nongeneralization of habituation occurs within the central neural circuit when two different afferent pathways are stimulated (Carew et al., 1971). Thus one might expect nongeneralization t o occur between the central and peripheral neural circuits since the afferent pathways are different. Peretz (1970) demonstrated that electrical stimulation of the ctendial nerve dishabituates the gill withdrawal response to tactile stimulation of a gill pinnule in a n isolated preparation. Recently, Lukowiak (unpublished observation) observed that tactile stimulation of the siphon dishabituates the gill response that had been habituated with repeated tactile stimulation of the pinnule. Thus activity in the central neural circuit conducted to the gill over the ctendial nerve appears to have a facilitatory influence on the peripheral neural circuit in the gill. An inhibitory influence of the central circuit on the peripheral neural circuit in the gill has also been demonstrated (Peretz and Howieson, 1973). Further details on the involvement of central and peripheral circuits in gill and siphon habituation may be found in a recent review (Jacklet and Lukowiak, 1974). Our results demonstrate that each neural circuit that mediates siphon behavior can modulate the response mediated by the other neural circuit. Thus, a stimulus to the siphon of the intact animal may evoke a response directly by the peripheral circuit but, additionally, information is sent to the central nervous system which may mediate a n additional withdrawal depending upon the state of the system. Also, responses mediated by central neurons may be modulated by the peripheral circuit depending upon its state. There are three sites of habituation, one in periphery between receptor and effector, one in the central reflex pathway and one at the peripheral terminals of the central projections. The central circuit may be responsible for long-term retention of habituation (Carew, Pinsker and Kandel, 1972) although it has been found that there is no difference in retention of short-term habituation between preparations with or without the central neurons (Lukowiak, 1973). Spontaneous contractions of the siphon and gill have been previously reported for intact and semi-intact preparations and attributed to the
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activity of neurons in the abdominal ganglion (Kupfermann and Kandel, 1969; Peretz, 1969). We have also seen abdominal ganglion neuron activity correlated with spontaneous contractions (Lukowiak, 1973) and a burst of activity in the siphon nerve preceding spontaneous contractions. Additionally, spontaneous contractions occur in the isolated siphon. Spontaneous contractions do not influence habituation of evoked responses in the isolated siphon or reflex contractions of the gill (Pinsker et al., 1970) but they may infrequently dishabituate gill pinnule responses (Peretz, 1970). Histological examination of the branch points of the siphon nerve in the siphon shows that there are many neurons, 10-50 p in diameter, in the nerve (Jacklet and Lukowiak, 1974). Also, these neurons have been selectively back-filled with cobalt through peripheral branches of the siphon nerve. These neurons along with other less clearly defined nerve net elements must mediate the siphon responses. Neurons distributed along peripheral nerve trunks are common in mollusks (Gorman and Mirolli, 1969; Prior, 1972a,b; Peretz and Estes, 1974), and a peripheral nerve net mediates responses (Hoyle and Willows, 1973). We propose a scheme for the peripheral neural circuit in the siphon that includes two receptors (tactile and photic) because the response latencies to tactile and light stimuli differ by a factor of 10 and tactile stimuli evoke an obvious burst of activity in the siphon nerve and light does not. There is evidence for mechanoreceptors and photoreceptors on the siphon of gastropods (Dijkgraaf, 1935; Bailey and Laverack, 1966; Millott, 1968; Crisp, 1972; Newby, 1973). The receptors synapse on peripheral neural elements and, additionally, send tactile information directly to the CNS. The peripheral neural plexus (PNP) must include motor neurons as well. Evidence for chemical synapses in the P N P is reported by Newby (1973) in the sensitivity of the light response to many presumed transmitter substances and the block of responses by high magnesium. The actual mechanisms of habituation and dishabituation are unknown but they may include chemical synaptic mechanisms such as homosynaptic depression and heterosynaptic facilitation (Kandel and Spencer, 1968; Kandel, Castellucci, Pinsker and Kupfermann, 1970; Carew et al., 1971; Kandel, 1974) that could operate at the receptor-neuron synapse or the neuromuscular synapse. The central neurons may terminate on the P N P and siphon musculature since this is shown to be a site of habituation and dishabituation. Adaptive siphon behavior is influenced by the state of the peripheral and central neural circuits, so any complete analysis of the neural correlates of this response must take into account the peripheral as well as the central sites of plasticity. This research was supported by National Institutes of Health Grant NS 08443 to
J.W.J.
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Accepted for publication August 1, 1974
