| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California, Irvine, California 92697-4550
Submitted 3 March 2004; accepted in final form 6 May 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
), and 5-HT is released in the hemolymph and the CNS in response to sensitizing stimuli (Levenson et al. 1999; Marinesco and Carew 2002). Moreover, sensory neuron (SN) to motor neuron (MN) synapses are facilitated during sensitization and provide a cellular correlate for this form of memory (Castellucci and Kandel 1976). Exogenous 5-HT application in vitro is sufficient to produce SN-MN facilitation, and 5-HT receptor antagonists can block this effect (Brunelli et al. 1976; Mercer et al. 1991). Moreover, in the preceding paper, we showed that sensitizing stimuli activate a large number of serotonergic neurons within the Aplysia CNS (Marinesco et al. 2004). However, the behavioral effects of such an increase in serotonergic tone are still poorly understood.
For example, it is not known whether 5-HT release in the CNS is, by itself, sufficient to sensitize defensive reflexes in freely moving animals. Indeed, although 5-HT clearly facilitates SN-MN synapses, it has also been shown to inhibit the siphon-withdrawal reflex in vitro (Fitzgerald and Carew 1991). Moreover, although 5-HT is known to be important for locomotion (Mackey and Carew 1983; McPherson and Blankenship 1992; Palovcik et al. 1982; Parsons and Pinsker 1989) and the regulation of heart rate (Koester et al. 1974; Liebeswar et al. 1975), it is not known whether increased 5-HT release in the CNS can enhance locomotion and/or heart rate in intact animals. In this study, we sought to mimic the generalized activation of the serotonergic system observed after noxious stimulation by administering the rate-limiting 5-HT precursor 5-hydroxytryptophan (5-HTP) to freely moving animals. Because most serotonergic neurons are tonically active, this treatment resulted in increased tonic 5-HT release in the CNS, revealed by facilitation of SN-MN synapses and increased firing rate of serotonergic neurons in the CNS. Animals treated with 5-HTP showed the behavioral signs of a defensive arousal response characterized by increased heart rate and locomotion. This same treatment, however, produced net inhibition of the tail-induced siphon-withdrawal reflex. It is possible that this effect was mediated by 5-HTP-induced inhibition of polysynaptic pathways between SNs and MNs, which functionally counteracted the 5-HTP-induced facilitation of SN-MN synapses. Therefore although 5-HT release certainly contributes to sensitization of defensive reflexes, it cannot be considered as solely sufficient for the expression of this form of memory.
Overall our data indicate that activation of the serotonergic system contributes both to the arousal response triggered by noxious stimulation (by increasing locomotion and heart rate) and to the facilitation of SN-MN synapses. Insofar as these facilitatory synaptic effects contribute to memory encoding for sensitization, our data suggest that the Aplysia serotonergic system is in a position to ensure that aversive stimuli are efficiently remembered.
Some of the data in this paper have been presented in abstract form (Marinesco et al. 2003).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Wild-caught adult Aplysia californica (Marinus, Long Beach, CA) weighing 
250 g were used throughout this study. Animals were housed in tanks containing aerated artificial seawater (ASW) kept at 15°C and fed dried seaweed three times a week. For behavioral experiments involving measurements of heart rate or duration of defensive reflexes, parapodia were removed surgically to gain better access to siphon and heart contractions. Animals were anesthetized in ice-cold ASW for 1 h before surgery and left undisturbed during 
5 days before behavioral experiments were undertaken.
To prepare the tissue samples, animals were first anesthetized by injection of 150–200 ml of 0.35 M MgCl2 and immediately dissected for removal of the entire CNS without the buccal ganglion. The ganglia were then fixed in 0.4% glutaraldehyde for 30 s and desheathed in 50:50 ASW:0.35 M MgCl2 to expose the neurons. Ganglia were then kept in a recording chamber continuously perfused with ASW [which contained (in mM) 460 NaCl, 55 MgCl2, 11 CaCl2, 10 KCl, and 10 Tris]. In some experiments, the composition of ASW was changed to achieve 0 Ca2+ and high (3 times) Mg2+ concentrations [it contained (in mM) 295 NaCl, 10 KCl, 176 MgCl2 and 10 Trizma]. In each case, ASW was buffered to a pH of 7.6 before use.
Intracellular recordings
Neurons were impaled with intracellular glass micropipettes (5–15 M
) filled with 3 M KCl. Membrane potential was measured using an Axoclamp 2B amplifier (Axon instruments, Union City, CA), digitized with an ITC-16 AD-DA computer interface (Instrutech, Great Neck, NY), and recorded on computer file with a homemade software written with Igor Pro 4.03 (Wavemetrics, Lake Oswego, OR). To assess synaptic transmission between tail SNs and tail MNs, we recorded intracellularly from one tail SN and one tail MN connected by a synapse as already described in the preceding companion paper (Marinesco et al. 2004).
In one experiment, we recorded from serotonergic neurons that were prelabeled with 5,7-dihydroxytryptamine, using the same procedure as in the preceding paper (Marinesco et al. 2004). After intracellular recordings, the same cells were re-impaled with microelectrodes filled with Neurobiotin (5%, Vector Labs, Burlingame, CA) in 1 M KCl. Neurobiotin was injected ionophoretically for 45 min to 1 h (+5-nA pulses, 500-ms duration at 1 Hz) to trace their processes in the pedal ganglion and allow immunohistochemical confirmation of their serotonergic nature. Immunohistochemical labeling of 5-HT and Neurobiotin was performed according to the protocol described in the preceding paper (Marinesco et al. 2004).
Chronoamperometric detection of neuronal 5-HT release par performed using carbon-fiber microelectrodes implanted in the neuropil of pleural ganglia. A more complete description of the method is given in the preceding paper (Marinesco et al. 2004) as well as in an earlier study (Marinesco and Carew 2002).
Behavioral studies
Heart rate, locomotion, and duration of tail-induced siphon withdrawal were assessed in animals treated with 5-HTP or ASW. 5-HTP was diluted into 1 ml of ASW per 50 g body wt and administered at 200 mg/kg by intrahemocoel injections through the foot. Heart rate was measured in freely moving animals using an ultrasound stethoscope (Koven Vasculascope 500, Alliance Medical, Russelville, MO). Heart rate was assessed during several periods of 1 min or 30 s. These values were then averaged to compute the mean heart rate over a 15-min period. To measure locomotion, animals were placed in a circular arena (26.5 cm diam) divided into four quadrants. The number of lines crossed during a 5-min period was taken as an index of the locomotor activity of the animal. Crawling upward so that the animal's head was out of the water was considered one line crossing. The siphon-withdrawal reflex was evoked by a mild tactile stimulus applied to the tip of the tail with a water jet (Teledyne Water Pik, Fort Collins, CO). The duration of the siphon withdrawal was measured between water jet onset and the first signs of siphon relaxation.
Tail-shock was applied to freely moving animals with a suction electrode using one or two 2-s trains of current pulses spaced by 5 s (current pulses were 10 ms, 40 mA applied at 50 Hz through a constant current stimulus isolation unit connected with a S88 stimulator; Grass Medical Instruments, Quincy, MA). When studying the CNS in vitro, electrical stimulation was applied directly to the tail-nerve (P9) through a suction electrode, using one 2-s train of 5-ms, 20-V pulses at 40 Hz applied through a constant voltage stimulus isolation unit connected to a S88 Grass stimulator.
Statistics
Data were presented as means ± SE. Comparisons between two data groups were performed using the Student's t-test for equal or unequal variances as determined by the F test (significance level was P < 0.05). For comparisons among three or more data groups, we used an ANOVA followed by an LSD post hoc test (P < 0.05). Statistics software was the analysis tool-pack of Microsoft Excel 2000 and SPSS for Windows version 10.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
5-HTP is the rate-limiting precursor of 5-HT and specifically increases 5-HT synthesis and release as shown by chronoamperometric experiments (Fickbohm and Katz 2000; Marinesco and Carew 2002). Because, in the preceding paper, we found most serotonergic neurons in the Aplysia CNS to be tonically active (Marinesco et al. 2004), we expected 5-HTP to increase tonic 5-HT release throughout the animal.
We assessed 5-HT release evoked by tail-nerve shock in the pleural ganglia of 10 animals that had been previously treated with 5-HTP, trained (1 or 2 tail-shocks spaced by 5 s, see METHODS) and tested for defensive arousal or sensitization in behavioral experiments (see following text). 5-HT release was measured with carbon fiber microelectrodes implanted in the pleural ganglia, 2–3 h after injection of 5-HTP (200 mg/kg) or its vehicle (ASW). Microelectrodes used to measure 5-HT concentration can also detect 5-HTP, although with a lower sensitivity (5 times less than for 5-HT). To minimize a possible contamination of the electrochemical signal with 5-HTP, ganglia were perfused for 30 min before beginning the chronoamperometric recordings so that 5-HTP could be washed out of the preparation. Therefore 5-HTP interference in our chronoamperometric recordings seems unlikely. In animals treated with 5-HTP, 5-HT release evoked by tail-nerve shock was increased 25-fold compared with control animals (1641 ± 410 vs. 39 ± 8 nM, n = 5, P = 0.03, Fig. 1). Consistent with a previous study (Marinesco and Carew 2002), evoked 5-HT release was thus enhanced by this concentration of 5-HTP.
|
; Glanzman et al. 1989; Mackey et al. 1989; Marinesco and Carew 2002; Mercer et al. 1991). 5-HTP was applied onto the isolated pleural-pedal-cerebral ganglia at a concentration of 2 mM to mimic the concentration administered to freely moving animals. 5-HTP application alone produced an increase in SN excitability: the same depolarizing pulse that produced two action potentials in baseline conditions evoked an average of 4.6 spikes 30 min after 5-HTP (Fig. 2, A and B). SN excitability was enhanced within a few minutes of 5-HTP application and continued to increase over the course of the 30-min recording. The mean latency for the effect of 5-HTP was 13.4 ± 2.8 min. In addition, SN-MN synapses were facilitated to 256% of their baseline level 45 min after 5-HTP (Fig. 2, C and D). The latency of this effect was longer than for SN excitability: excitatory postsynaptic potentials (EPSPs) were evoked in the MN by stimulating the SN every 15 min. Under these conditions, the first test after 5-HTP application (15 min) was always within 20% of the mean baseline EPSP. Synaptic facilitation appeared after a delay of 36 ± 4 min (Fig. 2, C and D). This difference in latency between excitability measures and synaptic facilitation may reflect the diffusion and penetration of 5-HTP into the ganglia. Excitability increases are detected mostly at the SN cell bodies and could be mediated by 5-HTP facilitating 5-HT release from serotonergic fibers innervating the somata (Zhang et al. 1991). Synaptic facilitation, however, would require 5-HTP penetration into the synaptic neuropil (Bunge et al. 1997; Zhang et al. 2003).
|
|
), suggesting that 5-HT levels after 5-HTP administration reached or even exceeded those evoked by sensitization training. Moreover, the 5-HTP-induced increase in the background activity of 5-HT neurons (Fig. 3) strongly supports the view that serotonergic neurons can excite each other, which could serve to amplify the effects of transient sensitizing stimuli. Effects of 5-HTP on arousal and sensitization of defensive reflexes
We next examined the behavioral effects of an injection of 5-HTP (200 mg/kg) and compared them to those induced by tail-shock. We monitored locomotion, heart rate, and duration of the tail-elicited siphon-withdrawal reflex in different groups of animals. Animals treated with 5-HTP rapidly became active and started locomoting within the testing arena. 5-HTP never elicited inking or produced noticeable manifestations of behavioral compromise. The animals exhibited large repetitive pedal waves characteristic of escape locomotion and crawled over increased distances compared with control animals. Animals tended to crawl along the sides of the arena so that the actual distance they covered was 19.6 cm/line crossing (see METHODS). The number of lines crossed per 5 min was initially low (2 lines/5 min), reflecting the baseline exploratory activity elicited by moving the animals to the experimental arena 30 min before starting the experiment. In control animals injected with vehicle (ASW), the locomotion index fell to 0 after an additional 30 min, suggesting that most animals remained still within the same quadrant when left undisturbed. In these animals, tail-shock increased locomotion to 3.3 ± 0.5 lines/5 min (64 cm/5 min), reflecting an escape response to the noxious stimulus (Fig. 4A). By contrast, a few minutes after 5-HTP injection, the locomotion index increased steadily to reach 13 ± 0.9 lines/5 min (255 cm/5 min, Fig. 4A). Tail-shock did not further change the locomotion values in animals that had been treated with 5-HTP.
|
Finally, we examined whether 5-HTP alone could produce sensitization of the tail-elicited siphon-withdrawal reflex in the absence of a noxious stimulus (e.g., tail-shock). In control animals injected with ASW, tail-shock increased the duration of the tail-elicited siphon-withdrawal reflex to 176% of its baseline value, showing sensitization of the reflex (Fig. 5A). In animals treated with 5-HTP, however, siphon withdrawal was actually inhibited to 79% of its baseline value (P < 0.01, Fig. 5A). This inhibition was significant compared with postinjection values in vehicle-treated animals (P = 0.02, Fig. 5B). Tail-shock overcame this inhibition, bringing siphon-withdrawal duration to 111% of its preinjection baseline value (Fig. 5B). When siphon-withdrawal reflex duration was normalized to its preshock (i.e., postinjection) values, tail-shock produced similar effects in control and 5-HTP-treated animals (ANOVA followed by LSD post hoc test, not significant at all time points). However, there was a modest trend toward a longer enhancement of the reflex in control animals (significantly enhanced above baseline during 45 min after tail-shock) than in 5-HTP-treated animals (significantly enhanced above baseline during 30 min, ANOVA followed by LSD post hoc test, P < 0.05, Fig. 5C). When sensitization was assessed as the increase from postinjection baseline values to postshock values in both groups of animals, tail-shock produced the same relative change: +44–45% increase in siphon-withdrawal duration (Fig. 5C). These data suggest that because tail-shock produced similar reflex enhancement in control and 5-HTP-treated animals, the neuronal circuits responsible for sensitization were not desensitized or antagonized by 5-HTP.
|
Given the well-documented role of 5-HT in sensitization, it was surprising that potentiation of 5-HT synthesis and release by 5-HTP produced inhibition of siphon withdrawal. To confirm this result, we increased 5-HT levels in freely moving Aplysia by adding 5-HT (250 µM) in the ambient ASW. This procedure is known to dramatically enhance 5-HT concentration in the hemolymph (Levenson et al. 1999) and to activate CCAAT enhancer-binding protein, an immediate-early gene implicated in the consolidation of long-term facilitation in tail SNs (Alberini et al. 1994). Animals placed in 5-HT (250 µM) showed increased locomotion (data not shown) consistent with the behavioral arousal observed after 5-HTP administration. Moreover, the tail-induced siphon-withdrawal reflex was inhibited to about the same extent as by 5-HTP (-30%, P < 0.01; Fig. 6). To determine whether this inhibitory effect could be overcome by higher concentrations of 5-HT, we exposed another group of animals to 500 µM 5-HT in their ambient seawater. We still observed a slight decrease in siphon-withdrawal duration when 5-HT was present in the seawater. However, these animals showed obvious additional behavioral responses in response to such a high concentration of 5-HT, including secretion of a mucus layer that slowly covered their body. It is thus possible that in addition to increasing 5-HT concentrations in the hemolymph, this treatment might have an aversive component capable of interfering with defensive reflexes as well. In contrast, injection of 5-HTP did not produce any behavioral responses indicating that it might be aversive. These results support the view that persistently elevated 5-HT levels in Aplysia have an inhibitory effect on siphon-withdrawal reflex, confirming earlier studies by Fitzgerald and Carew (1991). Therefore increased 5-HT release in the Aplysia CNS, in itself, cannot be viewed as solely sufficient to produce behavioral expression of sensitization of defensive reflexes.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Mackey and Carew 1983; Walters et al. 1981) and increased heart rate (Dieringer et al. 1978) as well as sensitization of defensive reflexes (Carew et al. 1971; Pinsker et al. 1973). Several lines of evidence suggest that these defensive responses are regulated by 5-HT. 1) Animals injected with 5-HT show increased locomotion (Mackey and Carew 1983; Palovcik et al. 1982; Parsons and Pinsker 1989); 2) heart rate is under the excitatory control of the serotonergic neuron RBhe (Koester et al. 1974; Mayeri et al. 1974), and 5-HT in the hemolymph increases blood pressure and heart rate (Liebeswar et al. 1975); and 3) 5-HT is necessary for sensitization and its cellular correlate, facilitation of SN-MN transmission (Brunelli et al. 1976; Glanzman et al. 1989; Mercer et al. 1991). Noxious stimulation triggers the release of both central (Marinesco and Carew 2002) and circulating 5-HT (Levenson et al. 1999) but also gives rise to the release of other transmitters including Phe-Met-Arg-Phe-amide (FMRFamide) (Mackey et al. 1987) and acetylcholine (Wright and Carew 1995). It is therefore likely that the different aspects of defensive behavior triggered by tail-shock may also include contributions from other nonserotonergic mechanisms triggered by noxious stimulation. Serotonin and arousal
In this study, administration of 5-HTP, which increases 5-HT synthesis, resulted in an increase in tonic 5-HT release in the CNS revealed by an increase in the firing rate of serotonergic neurons in the pedal and abdominal ganglia, facilitation of SN-MN synapses, and an increase in evoked 5-HT release. Therefore 5-HTP treatment resulted in an increase in serotonergic tone in the absence of a noxious stimulation. We found that 5-HTP significantly increased heart rate and locomotion, two behavioral manifestations of defensive arousal in Aplysia (Dieringer et al. 1978). The fact that 5-HTP injection mimicked the effects of tail-shock on locomotion and heart rate suggests that a major function of the general activation of the Aplysia serotonergic system after noxious stimulation might be to trigger and coordinate the different aspects of defensive behavior. We should note that defensive arousal triggered by noxious stimulation is qualitatively different from food-induced arousal, which is characterized by feeding posture, biting, and increased heart rate (Dieringer et al. 1978; Jing and Gillette 1995). Interestingly, the serotonergic metacerebral cells are involved in food-induced arousal, contributing to the enhancement of the rate and magnitude of biting responses (Kupfermann and Weiss 1982; Rosen et al. 1989; Weiss et al. 1978). Thus it appears that different Aplysia serotonergic systems are involved in both appetitive and defensive aspects of arousal. After noxious stimulation, the generalized increase in firing rate in the serotonergic system that we described in the preceding paper (Marinesco et al. 2004) could be one of the neuronal mechanisms responsible for the coordinated behavioral responses underlying defensive arousal. This function is consistent with the role of the mammalian serotonergic system in arousal, stress, and wakefulness (Adell et al. 1997; Amat et al. 1998; Mc Ginty and Harper 1976). Such a role for 5-HT might have been conserved throughout evolution because of its crucial importance for adaptation and survival.
Differential effects of increased serotonergic tone on defensive reflexes
Interestingly, 5-HTP inhibited tail-induced siphon withdrawal, whereas escape locomotion was enhanced. Such an inhibition of defensive withdrawal reflexes could favor escape from noxious stimuli by suppressing movements not directly involved in locomotion. Indeed, full-body contractions and locomotion are often mutually exclusive in several marine mollusks including Clione limacina (Norekian and Satterlie 1996). Moreover, relaxation of the siphon could enhance water flow across the gill and thus favor increased respiration during escape locomotion.
After 5-HTP administration, the tail-induced siphon-withdrawal reflex was inhibited despite facilitation of tail SN-MN synapses and increased SN excitability, two major neuronal correlates of sensitization in Aplysia. A similar dissociation between cellular and behavioral measures of memory had already been described during habituation of the tail-induced siphon-withdrawal reflex (Stopfer and Carew 1996). Such dissociations support the view that sensitization is coded at multiple neuronal sites within the reflex circuits and not only at SN-MN synapses (see reviews by Barbas et al. 2003; Frost et al. 1988; Trudeau and Castellucci 1992, 1993). Indeed, 5-HT produces both facilitatory and inhibitory effects in the reflex circuit. Whereas 5-HT is known to facilitate monosynaptic SN-MN transmission (Brunelli et al. 1976; Glanzman et al. 1989; Mercer et al. 1991), it has also been shown to depress several polysynaptic pathways between SNs and MNs (Barbas et al. 2003; Bristol et al. 2001; Cleary et al. 1995; Fischer et al. 1997; Frost and Kandel 1995; Frost et al. 1988; Storozhuk and Castellucci 1999; Trudeau and Castellucci 1992, 1993; Xu et al. 1995). It is possible that, after 5-HTP treatment, 5-HT-induced inhibition of some of these polysynaptic pathways could have counteracted facilitation of SN-MN synapses, thereby producing net behavioral inhibition of the reflex.
Interestingly, tail-shock overcame 5-HTP-induced inhibition of the reflex and induced significant sensitization. It is unlikely that this reversal of 5-HTP inhibitory effects could result from the actions of 5-HT released by tail-shock. 5-HTP alone facilitated SN-MN synapses and increased SN excitability and the firing rate of serotonergic neurons to a similar (or even larger) extent than comparable cellular effects after tail-shock in control animals. These results suggest that 5-HTP probably increased tonic 5-HT release to a level comparable to the evoked 5-HT release after tail-shock. Although a quantitative assessment of the basal 5-HT concentrations after 5-HTP and after tail-shock would help clarify this issue, it seems that the behavioral inhibition observed after 5-HTP cannot be attributed to insufficient levels of 5-HT.
A more plausible interpretation of the observed reversal of 5-HTP effects is that tail-shock triggered the release of other, nonserotonergic, neurotransmitters or modulators within the reflex circuit. The importance of such nonserotonergic factors can be illustrated by L29 interneurons. L29s are nonserotonergic excitatory interneurons in the abdominal ganglion that convey up to 40% of the excitation triggered by a tactile stimulus on the tail to LFS siphon MNs (Fischer and Carew 1993; Hawkins and Schacher 1989; Kistler et al. 1985). L29
LFS synapses are inhibited by 5-HT, but during sensitization training, a subset of these cells fire in direct response to tail-shock, giving rise to posttetanic potentiation that overcomes 5-HT-induced inhibition (Bristol et al. 2001). Thus activity in the reflex circuit can counteract at least some inhibitory effects of 5-HT. Other factors important for sensitization might be nonserotonergic neuromodulatory molecules such as small cardiac peptide (Abrams et al. 1984). Overall, the reversal of 5-HTP-induced reflex inhibition by tail-shock suggests that 5-HT release might not be solely sufficient for the induction of sensitization but could require the synergistic action of other neurotransmitters or neuromodulators within the reflex circuit.
Finally, it is possible that 5-HT-induced inhibition recovers quickly after 5-HT release in contrast to facilitation of SN-MN synapses, which has been shown to persist 15–20 min and even up to several days after 5-HT application (Brunelli et al. 1976; Casadio et al. 1999; Cleary et al. 1998; Scholz and Byrne 1987; Sutton and Carew 2000). Because 5-HT release evoked in the CNS by sensitizing stimuli is quite short-lasting (30–40 s) (Marinesco and Carew 2002), it is possible that 5-HT first induces a transient net inhibition of the reflexes that diminishes after a few minutes to be later replaced by sensitization (i.e.: longer-lasting facilitation of SN-MN synapses). In support of this view, long-term sensitization observed 24 h after sensitizing stimuli, is correlated with long-term facilitation expressed at SN-MN synapses, whereas interneuronal connections inhibited by 5-HT appear unchanged after such a delay (Cleary et al. 1998). Moreover, transient inhibition has often been observed shortly after tail-shock, before sensitization is fully expressed (Mackey et al. 1987; Marcus et al. 1988; Wright et al. 1991), and could be mediated at least in part by 5-HT (Fitzgerald and Carew 1991). Therefore the immediate inhibitory effect of 5-HT on tail-induced siphon-withdrawal reflex, expressed in a context of increased escape locomotion, is not inconsistent with the important role of this amine in the induction of sensitization.
Serotonergic neurons can contribute to both arousal and memory
Collectively, the present study and the preceding paper (Marinesco et al. 2004) indicate that a large number of interconnected serotonergic neurons are activated by noxious stimulation and that this generalized serotonergic activation in intact animals is sufficient to produce both defensive arousal and facilitation of SN-MN synapses, which ultimately contribute to encoding memory for sensitization. An interesting feature of the distributed serotonergic network identified in this study is that some individual neurons appear to contribute both to the defensive arousal response and to the encoding of at least one component of sensitization, synaptic facilitation. A good example of identified neurons shared between arousal and memory circuits might be the CC3/CB1 cells. These neurons project extensively to abdominal serotonergic neurons such as RBhe and to a significant number of pedal serotonergic neurons through monosynaptic excitatory connections. When CC3/CB1 neurons fire in response to noxious stimulation, they likely contribute to spreading excitation to other serotonergic neurons involved in defensive arousal like parapodia-opener-phase-like cells or RBhe and lead to a global serotonergic response in the CNS. CC3s (CB1s) could therefore serve as an "aversive stimulus" detector and trigger the coordinated neurochemical processes underlying defensive behavior. A similar function is commonly attributed to mammalian noradrenergic neurons in the locus coeruleus that seem to respond best to novel or aversive stimuli (Sara and Segal 1991; Vankov et al. 1995; review in Foote et al. 1983). In addition to their role in arousal, exciting other serotonergic neurons, CC3/CB1 neurons have been directly implicated in sensitization. They fire in response to tail-shock and their intracellular activation can facilitate siphon SN-MN synapses (Mackey et al. 1989).
The fact that specific neurons in the Aplysia CNS can, in principle, contribute to both arousal and memory formation is reminiscent of studies of fear conditioning in mammals. For example, the amygdala, which is involved in the encoding of emotionally arousing stimuli, also contributes to memory consolidation (Davis and Whalen 2001; LeDoux 2000; McGaugh et al. 2000; Zald 2003). Likewise, the nucleus of the solitary tract is critically involved in the regulation of heart rate and cardiovascular tone, as well as vagal motor control, and contributes to the adaptive responses to stressful stimuli. Noradrenergic neurons in this nucleus have also been shown to participate in the encoding of fearful memories (Clayton and Williams 2000; Williams and McGaugh 1993; Williams et al. 2000). Finally, stress hormones like corticosterone and epinephrine, which were first identified for their role in the fight-or-flight response through the regulation of cardiovascular tone or glucose metabolism, also improve the consolidation of fear memories, probably by influencing central noradrenergic systems (reviewed in Clayton and Williams 2000; McGaugh and Roozendaal 2002; McIntyre et al. 2002; Roozendaal et al. 1999). Therefore it is possible that sharing neuronal elements between arousal and memory circuits is a common strategy conserved through evolution to ensure that emotionally arousing stimuli are readily remembered.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address reprint requests and other correspondence to: T. J. Carew, Department of Neurobiology and Behavior, CNLM, University of California, Irvine, CA 92697-4550. (E-mail: tcarew{at}uci.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Adell A, Casanovas J, and Artigas F. Comparative study in the rat of the actions of different types of stress on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacology 36: 735–741, 1997.[CrossRef][ISI][Medline]
Alberini CM, Ghirardi M, Metz R, and Kandel ER. C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia. Cell 76: 1099–1114, 1994.[CrossRef][ISI][Medline]
Amat J, Matus-Amat P, Watkins L, and Maier S. Escapable and inescapable stress differentially alter extracellular levels of 5-HT in the basolateral amygdala of the rat. Brain Res 812: 113–120, 1998.[CrossRef][ISI][Medline]
Barbas D, DesGroseillers L, Castellucci V, Carew TJ, and Marinesco S. Multiple serotonergic mechanisms contributing to sensitization in Aplysia: evidence of diverse serotonin receptor subtypes. Learn Mem 10: 373–386, 2003.
Bristol AS, Fischer TM, and Carew TJ. Combined effects of intrinsic facilitation and modulatory inhibition of identified interneurons in the siphon withdrawal circuitry of Aplysia. J Neurosci 21: 8990–9000, 2001.
Brunelli M, Castellucci V, and Kandel ER. Synaptic facilitation and behavioral sensitization in Aplysia: possible role for serotonin and cyclic AMP. Science 194: 1178–1181, 1976.
Bunge SA, Mauelshagen J, and Carew TJ. Reversal of relative thresholds for synaptic facilitation and increased excitability induced by serotonin and tail nerve stimulation in Aplysia sensory neurons. Neurobiol Learn Mem 67: 259–63, 1997.[CrossRef][ISI][Medline]
Carew TJ, Castellucci VF, and Kandel ER. An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aplysia. Int J Neurosci 2: 79–98, 1971.[Medline]
Casadio A, Martin KC, Giustetto M, Zhu H, Chen M, Bartsch D, Bailey CH, and Kandel ER. A transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis. Cell 99: 221–37, 1999.[CrossRef][ISI][Medline]
Castellucci V and Kandel ER. Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science 194: 1176–8, 1976.
Clayton EC and Williams CL. Adrenergic activation of the nucleus tractus solitarius potentiates amygdala norepinephrine release and enhances retention performance in emotionally arousing and spatial memory tasks. Behav Brain Res 112: 151–158, 2000.[CrossRef][ISI][Medline]
Cleary LJ, Byrne JH, and Frost WN. Role of interneurons in defensive withdrawal reflexes in Aplysia. Learn Mem 2: 133–151, 1995.
Cleary LJ, Lee WL, and Byrne JH. Cellular correlates of long-term sensitization in Aplysia. J Neurosci 18: 5988–5998, 1998.
Davis M and Whalen PJ. The amygdala: vigilance and emotion. Mol Psychiatry 6: 13–34, 2001.[CrossRef][ISI][Medline]
Dieringer N, Koester J, and Weiss K. Adaptive changes in heart rate of Aplysia californica. J Comp Physiol [A] 123: 11–21, 1978.[CrossRef]
Fickbohm DJ and Katz PS. Paradoxical actions of the serotonin precursor 5-hydroxytryptophan on the activity of identified serotonergic neurons in a simple motor circuit. J Neurosci 20: 1622–1634, 2000.
Fischer TM, Blazis DE, Priver NA, and Carew TJ. Metaplasticity at identified inhibitory synapses in Aplysia. Nature 389: 860–865, 1997.[CrossRef][Medline]
Fitzgerald K and Carew TJ. Serotonin mimics tail shock in producing transient inhibition in the siphon withdrawal reflex of Aplysia. J Neurosci 11: 2510–2518, 1991.[Abstract]
Fischer TM and Carew TJ. Activity-dependent potentiation of recurrent inhibition: a mechanism for dynamic gain control in the siphon withdrawal reflex of Aplysia. J Neurosci 13: 1302–1314, 1993.[Abstract]
Foote SL, Bloom FE, and Aston-Jones G. Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol Rev 63: 844–914, 1983.
Frost WN, Clark GA, and Kandel ER. Parallel processing of short-term memory for sensitization in Aplysia. J Neurobiol 19: 297–334, 1988.[CrossRef][ISI][Medline]
Frost WN and Kandel ER. Structure of the network mediating siphon-elicited siphon withdrawal in Aplysia. J Neurophysiol 73: 2413–2427, 1995.
Glanzman DL, Mackey SL, Hawkins RD, Dyke AM, Lloyd PE, and Kandel ER. Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock. J Neurosci 9: 4200–4213, 1989.[Abstract]
Hawkins RD and Schacher S. Identified facilitator neurons L29 and L28 are excited by cutaneous stimuli used in dishabituation, sensitization, and classical conditioning of Aplysia. J Neurosci 9: 4236–4245, 1989.[Abstract]
Hening WA, Walters ET, Carew TJ, and Kandel ER. Motorneuronal control of locomotion in Aplysia. Brain Res 179: 231–53, 1979.[CrossRef][ISI][Medline]
Jing J and Gillette R. Neuronal elements that mediate escape swimming and suppress feeding behavior in the predatory sea slug Pleurobranchaea. J Neurophysiol 1995: 1900–1910, 1995.
Kistler HB, Hawkins RD, Koester J, Steinbush HWM, Kandel ER, and Schwartz JH. Distribution of serotonin-immunoreactive cell bodies and processes in the abdominal ganglion of mature Aplysia. J Neurosci 5: 72–80, 1985.[Abstract]
Koester J, Mayeri E, Liebeswar G, and Kandel ER. Neural control of circulation in Aplysia: II Interneurons. J Neurophysiol 37: 476–496, 1974.
Kupfermann I and Weiss KR. Activity of an identified serotonergic neuron in free moving Aplysia correlates with behavioral arousal. Brain Res 241: 334–337, 1982.[CrossRef][ISI][Medline]
LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci 23: 155–184, 2000.[CrossRef][ISI][Medline]
Levenson J, Byrne JH, and Eskin A. Levels of serotonin in the hemolymph of Aplysia are modulated by light/dark cycles and sensitization training. J Neurosci 19: 8094–8103, 1999.
Liebeswar E, Goldman JE, Koester J, and Mayeri E. Neural control of circulation in Aplysia. III. Neurotransmitters. J Neurophysiol 38: 767–779, 1975.
Mackey SL and Carew TJ. Locomotion in Aplysia: triggering by serotonin and bag cells extract. J Neurosci 3: 1469–1477, 1983.[ISI][Medline]
Mackey SL, Glanzman DL, Small SA, Dyke AM, Kandel ER, and Hawkins RD. Tail-shock produces inhibition as well as sensitization of the siphon-withdrawal reflex of Aplysia: possible behavioral role for presynaptic inhibition mediated by the peptide Phe-Met-Arg-Phe-NH2. Proc Natl Acad Sci USA 84: 8730–8734, 1987.
Mackey SL, Kandel ER, and Hawkins RD. Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons. J Neurosci 9: 4227–4236, 1989.[Abstract]
Marcus EA, NolenTG, Rankin CH, and Carew, TJ. Behavioral dissociation of dishabituation, sensitization, and inhibition of Aplysia. Science 241: 210–213, 1988.
Marinesco S and Carew TJ. Serotonin release evoked by tail-nerve stimulation in Aplysia: characterization and relationship to heterosynaptic plasticity. J Neurosci 22: 2299–2312, 2002.
Marinesco S, Kolkman KE, Wickremasinghe N, and Carew TJ. Systemic elevation of serotonergic levels in freely-moving Aplysia inhibits tail-induced siphon withdrawal reflex. Soc Neurosci Abstr 520.1, 2003.
Marinesco S, Kolkman KE, and Carew TJ. Serotonergic modulation in Aplysia. I. Distributed serotonergic network persistently activated by sensitizing stimuli. J Neurophysiol 92: 2468–2486, 2004.
Mayeri E, Koester J, Kupferman I, Liebeswar G, and Kandel ER. Neural control of circulation in Aplysia. II. Motoneurons. J Neurophysiol 37: 458–475, 1974.
Mc Ginty DJ and Harper RM. Dorsal raphe neurons: depression of firing during sleep in cats. Brain Res 101: 569–575, 1976.[CrossRef][ISI][Medline]
McGaugh JL, Ferry B, Vazdarjanova A, and Roozendaal B. Amygdala role in modulation of memory storage. In: The Amygdala: A Functional Analysis, edited by Aggleton JP. Oxford, UK: Oxford Univ. Press, 2000, p. 391–424.
McGaugh JL and Roozendaal B. Role of adrenal stress hormones in forming lasting memories in the brain. Curr Opin Neurobiol 12: 205–210, 2002.[CrossRef][ISI][Medline]
McIntyre CK, Hatfield T, and McGaugh J. Amygdala norepinephrine levels after training predict inhibitory avoidance retention performance in rats. Eur J Neurosci 16: 1223–1226, 2002.[CrossRef][ISI][Medline]
McPherson DR and Blankenship JE. Neuronal modulation of foot and body-wall contractions in Aplysia californica. J Neurophysiol 67: 23–28, 1992.
Mercer AR, Emptage NJ, and Carew TJ. Pharmacological dissociation of modulatory effects of serotonin in Aplysia sensory neurons. Science 254: 1811–1813, 1991.
Norekian TP and Satterlie RA. Cerebral serotonergic neurons reciprocally modulate swim and withdrawal neural networks in the mollusk Clione limacina. J Neurophysiol 75: 538–546, 1996.
Palovcik RA, Basberg BA, and Ram JL. Behavioral state changes induced in Pleurobranchaea and Aplysia by serotonin. Behav Neural Biol 35: 383–394, 1982.[CrossRef][ISI][Medline]
Parsons DW and Pinsker HM. Swimming in Aplysia brasiliana: behavioral and cellular effects of serotonin. J Neurophysiol 62: 1163–1176, 1989.
Pinsker HM, Hening WA, Carew TJ, and Kandel ER. Long-term sensitization of a defensive withdrawal reflex in Aplysia. Science 182: 1039–1042, 1973.
Roozendaal B, Nguyen BT, Power AE, and McGaugh JL. Basolateral amygdala noradrenergic influence enables enhancement of memory consolidation induced by hippocampal glucocorticoid receptor activation. Proc Natl Acad Sci USA 96: 11642–11647, 1999.
Rosen SC, Weiss KR, Goldstein RS, and Kupfermann I. The role of a modulatory neuron in feeding and satiation in Aplysia: effects of lesioning of the serotonergic metacerebral cells. J Neurosci 9: 1562–1578, 1989.[Abstract]
Sara SL and Segal M. Plasticity of sensory responses of locus coeruleus neurons neurons in behaving rats: implication for cognition. Prog Brain Res 88: 457–465, 1991.
Scholz KP and Byrne JH. Long-term sensitization in Aplysia: biophysical correlates in tail sensory neurons. Science 235: 685–687, 1987.
Stopfer M and Carew TJ. Heterosynaptic facilitation of tail sensory neuron synaptic transmission during habituation in tail-induced tail and siphon withdrawal reflexes of Aplysia. J Neurosci 16: 4933–48, 1996.
Storozhuk M and Castellucci VF. Modulation of cholinergic transmission in the neuronal network of the gill and siphon withdrawal reflex in Aplysia. Neuroscience 90: 291–301, 1999.[CrossRef][ISI][Medline]
Sutton MA and Carew TJ. Parallel molecular pathways mediate expression of distinct forms of intermediate-term facilitation at tail sensory-motor synapses in Aplysia. Neuron 26: 219–31, 2000.[CrossRef][ISI][Medline]
Trudeau LE and Castellucci VF. Contribution of polysynaptic pathways in the mediation and plasticity of Aplysia gill and siphon withdrawal reflex: evidence for differential modulation. J Neurosci 12: 3838–3848, 1992.[Abstract]
Trudeau LE and Castellucci VF. Sensitization of the gill and siphon withdrawal reflex of Aplysia: multiple sites of change in the neuronal network. J Neurophysiol 70: 1210–1220, 1993.
Vankov A, Herve-Minvielle A, and Sara S. Response to novelty and its rapid habituation in locus coeruleus neurons of the freely exploring rat. Eur J Neurosci 7: 1180–1187, 1995.[CrossRef][ISI][Medline]
Walters ET, Carew TJ, and Kandel ER. Associative Learning in Aplysia: evidence for conditioned fear in an invertebrate. Science 211: 504–6, 1981.
Weiss KR, Cohen J, and Kupferman I. Modulatory control of buccal musculature by a serotonergic neuron (metacerebral cell) in Aplysia. J Neurophysiol 41: 181–203, 1978.
Williams CL and McGaugh JL. Reversible lesions of the nucleus of the solitary tract attenuate the memory-modulating effects of posttraining epinephrine. Behav Neurosci 107: 1–8, 1993.
Williams CL, Men D, and Clayton EC. The effects of noradrenergic activation of the nucleus tractus solitarius on memory and in potentiating norepinephrine release in the amygdala. Behav Neurosci 114: 1131–1144, 2000.[CrossRef][ISI][Medline]
Wright WG and Carew TJ. A single identified interneuron gates tail-shock induced inhibition in the siphon withdrawal reflex of Aplysia. J Neurosci 15: 790–797, 1995.[Abstract]
Wright WG, Marcus EA, and Carew TJ. A cellular analysis of inhibition in the siphon withdrawal reflex of Aplysia. J Neurosci 11: 2498–2509, 1991.[Abstract]
Xu Y, Pieroni JP, Cleary LJ, and Byrne JH. Modulation of an inhibitory interneuron in the neural circuitry for the tail withdrawal reflex of Aplysia. J Neurophysiol 73: 1313–1318, 1995.
Zald DH. The human amygdala and the emotional evaluation of sensory stimuli. Brain Res Rev 41: 88–123, 2003.[CrossRef][Medline]
Zhang H, Wainwright M, Byrne JH, and Cleary LJ. Quantitation of contacts among sensory, motor, and serotonergic neurons in the pedal ganglion of Aplysia. Learn Mem 10: 387–93, 2003.
Zhang ZS, Fang B, Marshak DW, Byrne JH, and Cleary LJ. Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia. J Comp Neurol 311: 259–70, 1991.[CrossRef][ISI][Medline]
This article has been cited by other articles:
|
|
 |
|
  X. Ye, J. L. Shobe, S. K. Sharma, A. Marina, and T. J. Carew Small G proteins exhibit pattern sensitivity in MAPK activation during the induction of memory and synaptic facilitation in Aplysia PNAS, December 23, 2008; 105(51): 20511 - 20516. [Abstract] [Full Text] [PDF]
|
|
). Exogenous 5-HT inhibited siphon-withdrawal reflex to the same extent as 5-HTP (n = 9). *, P < 0.05.