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1Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan; and 2Department of Biology, University of Washington, Seattle Washington
Submitted 13 May 2005; accepted in final form 22 June 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Stimuli applied to the tailfan are detected by external mechanosensory hairs and internal proprioceptors. LG receives these extero- and proprioceptive inputs as excitatory postsynaptic potentials (EPSPs) directly via electrical (
component) and chemical synapses (' component) from the afferents and indirectly through sensory interneurons (
component) (Araki and Nagayama 2003; Newland et al. 1997; Wine and Krasne 1972). The LGs are known to habituate readily after repeated sensory stimulation as a result of a decline in the efficacy of chemically mediated synaptic transmission from sensory afferents to LG or sensory interneurons (Araki and Nagayama 2003, 2005; Zucker 1972).
Serotonin (5-hydroxytryptamine; 5-HT) and octopamine are neuroactive monoamines known to affect feeding, sexual, postural, and aggressive behaviors in both vertebrates and invertebrates (Antonsen and Paul 1997; Lesch and Merschdorf 2000; Livingstone et al. 1980; Weiger 1997) and have a modulatory action on neural activity underlying learning and memory in many invertebrates (Hammer and Menzel 1998; Kandel 2001; Schwaerzel et al. 2003). In the crayfish and the lobster, neurons that contain 5-HT and octopamine are widely distributed in the CNS (Real and Czternasty 1990; Schneider et al. 1993, 1996). 5-HT and octopamine act as physiological regulators to enhance or reduce the responsiveness of central and peripheral synapses (Djokaj et al. 2001; Kravitz and Huber 2003; Livingstone et al. 1980; Nagayama 2002), including, for example, modulation of the neural circuit for the LG-mediated tailflip. Octopamine enhances the synaptic response of LGs during sensory stimulation (Bustamante and Krasne 1991; Glanzman and Krasne 1983), whereas 5-HT can enhance or depress the LG response, depending on the social status of an animal (Yeh et al. 1996, 1997) or dose, rate, and time course of exposure (Teshiba et al. 2001).
All 5-HT and octopamine receptors, with the exception of 5-HT3 receptor in which activation leads to quick opening of ion channel, belong to a superfamily of G-protein-coupled receptors, and their effects are mediated by second messengers (Hoyer et al. 1994; Roeder 1999). Both 5-HT and octopamine have been shown to play a crucial role in associative learning by increasing cyclic adenosine monophosphate (cAMP) levels in a siphon withdrawal reflex of Aplysia (Kandel 2001) and in a proboscis extension reflex of honeybee (Hammer and Menzel 1998). Furthermore, in mammals, activation of the 5-HT2B receptor increases cyclic guanosine monophosphate (cGMP) levels (Manivet et al. 2000). Thus the modulatory effects of 5-HT and octopamine on the neural circuit for LG-mediated tailflips must be related to a second-messenger cascade. Only Edwards et al. (2002) have demonstrated, using bath application of 50 µM dibutyrl cAMP, that the amplitude of LG EPSPs to sensory stimulation is facilitated. However, the second-messenger mechanisms stimulated by 5-HT and octopamine are still largely unknown. Furthermore, there is only a small body of evidence that shows that 5-HT and octopamine modulate the process of LG habituation (Araki and Nagayama 2005).
In this study, using bath application of 5-HT and octopamine at low concentrations (5–10 µM), we have analyzed the effect of 5-HT and octopamine on the response of LGs to repetitive sensory stimulation. Their modulatory effect on LG habituation was compared with effects of cAMP and cGMP analogues. The results showed that both 5-HT and octopamine increased the number of stimuli necessary to cause LG habituation and that only cAMP mimicked these effects. A 5-HT-induced enhancement of the LG responsiveness to sensory stimulation was mediated by cAMP but the effect of octopamine was cAMP-independent.
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METHODS |
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Adult male and female crayfish, Procambarus clarkii Girard (6–9 cm body length from rostrum to telson) were used in all experiments. Crayfish were purchased commercially from a local supplier and maintained in laboratory fresh water tanks and fed weekly on a diet of chopped potato and liver. Prior to experiments, crayfish were isolated individually in small tanks (20 x 35 x 25 cm) for 
1 wk. There were no significant differences in results according to sex and body length.
The nerve chain from the 2nd to terminal (6th) abdominal ganglion with relevant nerve roots was isolated from the abdomen and pinned, dorsal-side-up, in a silicone elastomer (Sylgard)-lined perfusion chamber, containing cooled van Harreveld's (1936) solution. The dorsal ganglionic sheath of the terminal ganglion was surgically removed with fine forceps to facilitate drug perfusion and microelectrode penetration.
The spike activity of LG was monitored extracellularly from the fourth to fifth abdominal connective using a suction electrode. Nerve roots 2–4 of the terminal abdominal ganglion, which contain mechanosensory afferents innervating the uropods and telson, were electrically stimulated simultaneously using a single oil hook electrode. Square pulses (0.01- to 0.05-ms duration; 1- to 20-V intensity) were delivered through the stimulating electrode.
Habituation curve
After 15 min rest after dissection, the spike threshold of LG to sensory stimulation was determined by gradually increasing the intensity of stimulation of the sensory nerves with a 5-s interstimulus interval. After the LG spike threshold was determined, the intensity of stimulation was set so that the stimulus was just suprathreshold. The preparation was rested for a further 5 min before repeated sensory stimulation was applied with an inter-stimulus interval of 5 s until the LG failed to give rise to spikes after five continuous trials. Preparations that did not show habituation after 40 trials of stimulation were counted as "censored" preparations. The spike rate of the LGs was calculated by averaging each trial of stimulation and was shown as a habituation curve.
The following pharmacological agents were obtained from Sigma: 5-HT, octopamine, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, cAMP analogue), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, cGMP analogue), and 3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase inhibitor). Adenylate cyclase inhibitor, SQ22536 was obtained from Calbiochem. These agents were all dissolved in normal saline to concentrations of 10 µM 5-HT, 10 µM octopamine, mixture of 50 µM CPT-cAMP and 50 µM IBMX, 100 µM 8-Br-cGMP and 100 µM SQ22536. For bath application of these agents, the perfusion chamber was 4.0 ml in volume, and the bathing saline, with and without drugs, was supplied with a microtube pump (Eyla, MP-3) at a rate of 4.5 ml/min. After the stimulus intensity was set, one of these drugs, exclusive of 100 µM SQ22536, was perfused for 5 min, then washed out with normal saline. Just after washing with normal saline, repetitive sensory stimulation was applied. In some preparations, 100 µM SQ22536-contained saline was continuously perfused for 15 min after dissection, then 10 µM 5-HT or 10 µM octopamine was co-applied with SQ22536 for 5 min after set of stimulus intensity. The differences in stimulus numbers needed to habituate LG were analyzed statistically using a log rank test.
Intracellular recordings
Intracellular recordings were made in the left half of the terminal ganglion neuropil with glass microelectrodes filled with 2 M potassium acetate. Electrode resistance was between 20 and 45 M
. LG identity was confirmed physiologically by a one-to-one correspondence of spikes between intracellular and extracellular recordings.
After successful recording from the LG, the stimulus intensity of sensory nerves was set subthreshold to elicit LG spike. The preparation was then rested for 5 min with bath application of 5 µM 5-HT, 10 µM octopamine, or 50 µM CPT-cAMP with 50 µM IBMX. In some preparations, 100 µM SQ22536-contained saline was continuously perfused for 
15 min before bath application of 5 µM 5-HT or 10 µM octopamine. Then the sensory stimulation at the same intensity was applied to observe the synaptic response of LGs. After stimulation, the preparation was washed with normal saline for 
20 min. In other preparations, the cAMP analogue resistant to phosphodiesterase, Sp-isomer of adenosine-3',5'-cyclic monophosphorothioate (Sp-cAMPS, Biolog) was directly injected into the LGs using 50 mM Sp-cAMPS (diluted in 0.5 M potassium chloride) filled glass microelectrodes (electrode resistance was between 40 and 80 M). Sp-cAMPS was injected iontophoretically with the use of 1- to 2-nA hyperpolarizing current pulses 500 ms in duration at 1 Hz for 5 min. Sensory stimulation evokes compound EPSPs in LG with at least three components. The first and second components (termed the and ' components) occur with a short latency. They are mediated through monosynaptic electrical and chemical synapses from mechanosensory afferents. The third component, the component, occurs with a longer latency and is mediated through a disynaptic pathway involving many intersegmental ascending interneurons (Araki and Nagayama 2003; Zucker 1972). We measured the peak amplitude of sensory-evoked EPSP as early (+' components) and later parts ( component) respectively, because the later EPSP, the component, could be readily discriminated from the early components, but separation of the and ' components was temporally difficult. In some cases, the amplitude of the component was enhanced to generate a spike after drug application. In such cases, the EPSP just prior to spike generation was estimated as peak amplitude of the component. The effect of drug application was analyzed statistically using repeated-measures ANOVA.
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RESULTS |
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EFFECT OF 5-HT AND OCTOPAMINE.
Sensory stimulation applied to second to fourth nerve roots of the terminal abdominal ganglion gave rise to a spike in the LG interneuron. When the stimulus was applied repeatedly, at an interstimulus interval of 5 s, the response of LG decreased gradually and showed habituation (Fig. 1). In control tests, the LG response readily showed a habituation, decreasing by >50% within five trials of sensory stimulation (Fig. 1, 
). The response declined by 80% after 20 trials, and only in 10% of preparations did it not show habituation after the 40th stimulus. Under bath application of 10 µM 5-HT (Fig. 1, 
) and 10 µM octopamine (Fig. 1, 
), the rate of decease in the response of LG to stimulation was slower. Only 20–35% of the preparations failed to respond with a spike within five trials of stimulation. The response of LG decreased by 50% just after 15 stimuli, and 30% of the preparations still responded with a spike after 40 stimuli. Thus both 5-HT and octopamine decreased the rate of habituation and the numbers of stimuli needed to habituate LG increased significantly (P < 0.05; log rank test).
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It is well known that 5-HT and octopamine stimulate intracellular messenger cascades (see for review Roeder 1999; Weiger 1997). To examine this possibility, the effects of cyclic nucleotide analogues on LG habituation were examined (Fig. 2). The rate of decrease in the LG response to repeated sensory stimulation became significantly slower after bath application of 50 µM CPT-cAMP (cAMP analogue) with 50 µM IBMX (cAMP phosphodiesterase inhibitor) (Fig. 2, 
). About 70% of the preparations did not show habituation even after the 15th stimulus, whereas >50% of the preparations still responded with a spike after 40 stimuli. As compared with control (Fig. 2, ), the cAMP analogue decreased the rate of habituation significantly (P < 0.001; log rank test). By contrast, bath application of 100 µM 8-Br-cGMP had no significant effect (Fig. 2, 
). Habituation curve of cGMP applied preparations was very similar to that of control. Log rank test showed no significant difference in stimulus numbers needed to habituate between control and cGMP applied preparations (P > 0.4). Thus only cAMP had similar inhibitory effect with that of 5-HT and octopamine.
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) between control (Figs. 1 and 3A, ) and preparations in which 5-HT was applied under normal saline (Figs. 1 and 3A, ). For later stimuli, the decline of the habituation curve of 5-HT applied with SQ22536 was similar to that of control. The effect of 5-HT under SQ22536 was not statistically different from the control (P > 0.9 between control and 5-HT/SQ22536-contained saline; log rank test). The number of stimuli needed to reach the onset of habituation in each preparation was plotted to show differences in distribution (Fig. 3B). In control, 70% of preparations showed habituation within 10 trials (Fig. 3B, bottom), and only 10% of preparations did not show habituation within 40 trials (censored preparations; indicated by Fig. 3B, ). The median stimulus number to habituate was 3 (
). In the 5-HT-applied group under normal saline, the number of preparations habituated within 10 trials decreased to 50% and censored preparations increased 35% (Fig. 3B, top). The median stimulus numbers to reach the onset of habituation was 12.5. When SQ22536 was continuously perfused before bath application of 5-HT, 60% of preparations habituated within 10 trials (Fig. 3B, middle). In this group, the number of preparations that did not show habituation reduced to 5%, and the median number to habituate decreased to 6. Bath application of SQ22536 itself had no significant effect on the responsiveness of the LGs because the stimulus intensity for spike threshold of LGs was similar between control (10.5 ± 0.9 V; mean ± SE; n = 34) and SQ22536-applied preparations (10.4 ± 0.9 V; n = 36).
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and ), and no significant difference was observed statistically (P > 0.25 between octopamine/normal saline and octopamine/SQ22536-contained saline; long rank test). The distribution of the stimulus number needed to reach the onset of habituation was also similar between octopamine with and without preincubation of SQ22536 (Fig. 3D). The median stimulus number to reach the onset of habituation was 19.5 in the octopamine-applied group under normal saline, and 20 in octopamine-applied group under SQ22536-containing saline. Compared with control, quickly habituated preparations were smaller in number and preparations that did not show habituation increased in the octopamine/SQ22536 group. The number of trails to habituate was significantly higher than that of control (P < 0.05 between control and octopamine/SQ22536-contained saline; log rank test). These results suggest that the signaling pathway of octopamine is cAMP independent. Enhancement of synaptic response of LGs
EFFECT OF 5-HT AND OCTOPAMINE.
Bath application of 5 µM 5-HT had no strictly significant effect on the resting membrane potential of LGs. The mean resting membrane potential of LGs was –84.3 ± 1.0 (SE) mV (n = 40) in normal saline and resting potential was slightly depolarized in amplitude (0.5 ± 0.7 mV; n = 7) 5 min after 5-HT application. The sensory-evoked EPSP of LG was, by contrast, increased in amplitude, including both the early +''component and the later component (Fig. 4 Ai, middle). The amplitude of EPSP of +' component increased to 110% (107.6 ± 3.4%; n = 7) compared with the initial amplitude (Fig. 4A, i and ii, top). The amplitude of component also increased to 120% (117.8 ± 6.2%). The increase in amplitude of both components was statistically significant (P < 0.05 in both +' component and component; repeated-measures ANOVA; Fig. 4C). The amplitude of sensory-evoked EPSP was significantly smaller when the sensory stimulation was applied 5 min after a pause without 5-HT application (control in Fig. 4C). Thus the bath application of 5-HT enhanced the synaptic response of LGs to the sensory stimulation. The response of LG recovered partially after 20 min of washing with normal saline (Fig. 4Ai, bottom).
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+' component after 5-HT application was slightly increased by 0.7 ± 1.3% (mean ± SE: n = 5) and that of component by only 2.2 ± 3.5% (P > 0.8 for the +' component and P > 0.6 for the components; repeated-measures ANOVA; Fig. 4C).
Bath application of 10 µM octopamine also increased the amplitude of sensory-evoked EPSPs of the LGs that frequently induced a spike (Fig. 5 A). The resting membrane potential of the LGs showed a slight depolarization (0.3 ± 1.0 mV; n = 7) 5 min after octopamine application. The amplitude of the early +' component and later component increased to 120% (115.2 ± 5.1%) and 120% (121.7 ± 8.6%) of the initial EPSP, respectively (Fig. 5C). Both increases were statistically significant (P < 0.05 in both +' component and component; repeated-measures ANOVA). The response of LG to sensory stimulation partly recovered after 20-min washing (not shown). In contrast with 5-HT application, preincubation of 100 µM SQ22536 prior to bath application of octopamine did not suppress the enhanced effect of octopamine on the LG response (Fig. 5B). The resting membrane potential of the LGs after bath application of octopamine under SQ22536 was slightly shifted negatively (–2.1 ± 1.2 mV; n = 6). The amplitude of EPSP of early +' component increased by 12.9 ± 3.0% and that of component increased by 28.2 ± 7.0% (Fig. 5C) and were significantly larger than sensory-evoked EPSP before octopamine application (P < 0.01 in both +' and components; repeated-measures ANOVA).
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The intracellular cAMP level is increased by activation of adenylate cyclase. The 5-HT-induced synaptic enhancement of LGs is likely to be mediated by cAMP because the effect of 5-HT was canceled under continuous perfusion of SQ22536 that inhibited the activation of adenylate cyclase. A co-application of cAMP analogue, CPT-cAMP of 50 µM, and the phosphodiesterase inhibitor, IBMX of 50 µM were applied to investigate this possibility (Fig. 6 A).
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+' component and the later component, and the LGs responded with a spike 5 min after CPT-cAMP application (Fig. 6A). The peak amplitude of the +' component increased by 6.7 ± 2.0% and that of the component by 13.3 ± 3.1% (n = 5); both increases were statistically significant (P < 0.05 for the +' component and P < 0.01 for the component; repeated-measures ANOVA). The synaptic enhancement of LG to sensory stimulation was still observed after 20 min of washing with normal saline (not shown).
Bath application of cAMP analogue, CPT-cAMP, could affect various neural elements within the terminal abdominal ganglion. To investigate the action of cAMP on LG, we iontophoretically injected Sp-cAMPS, an cAMP analogue resistant to phosphodiesterase (Hochner and Kandel 1992; Otmakhov and Lisman 2002), directly into LG using the recording glass microelectrodes (Fig. 6B). Sp-cAMPS of 50 mM was diluted in 0.5 M KCl solution and injected into LG by applying 1- to 2-nA, 500-ms hyperpolarizing current pulse at 1 Hz for 5 min. After intracellular injection of Sp-cAMPS, the resting membrane potential of the LGs showed a small membrane depolarization (5.4 ± 1.8 mV; n = 11). This membrane depolarization is likely to be due to an increase in the level of internal cAMP since the resting membrane potential was unchanged (–0.1 ± 0.1 mV; n = 5) 5 min after hyperpolarizing current injection using 0.5 M KCl filled electrode without Sp-cAMPS. Both the early +' component and the later component of the sensory-evoked EPSP of the LGs were increased in amplitude after intracellular injection of Sp-cAMPS. The group mean amplitude of the +' component increased by 19.1 ± 4.2% (n = 11) and that of the component by 20.4 ± 6.2% (Fig. 6C). The difference of EPSP amplitude before and after Sp-cAMPS injection was statistically significant (P < 0.001 for the +' component and P < 0.01 for the components; repeated-measures ANOVA).
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DISCUSSION |
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Rate of habituation
Both 5-HT and octopamine decreased the rate of LG habituation to repetitive sensory stimulation and bath application of a cAMP analogue showed a similar decrement in the habituation rate. Because the excitability of LG to sensory stimulation was enhanced by bath application of 5-HT, octopamine and the cAMP analogue, then the sensory-evoked EPSPs were increased in amplitude. The amount of habituation per stimulus decreases with increasing stimulus strength (Christoffersen 1997), therefore the enhanced response of LG to sensory stimulation by 5-HT, octopamine, and cAMP decreased the rate of habituation.
In many cases, 5-HT is known to have antagonistic effect with octopamine. For example, in lobster and crayfish, the injection of octopamine leads to a rigidly extended posture while injection of 5-HT induces a rigid body flexion (Livingstone et al. 1980). The modulatory effect of 5-HT on the synaptic response of LGs, depends on the social status of an animal (Yeh et al. 1996, 1997), or dose, rate, and time course of 5-HT exposure (Glanzman and Krasne 1983; Teshiba et al. 2001). Although application of low concentrations of 5-HT facilitated the LG response to sensory stimulation, the effect was exposure-dependent when higher concentration of 5-HT was applied (Edwards et al. 2002; Teshiba et al. 2001). A fast rate exposure of 5-HT inhibited the LG response, whereas a slow rate exposure facilitated the LG response. We focused on the effect of 5-HT of 5–10 µM concentration on LG excitability. The sensory-evoked EPSPs of LGs consistently increased in amplitude under bath-application of this concentration of 5-HT. We have not examined, in this paper, the effect of 5-HT at higher concentrations.
5-HT-cAMP cascade
Bath application of 5 µM 5-HT and 10 µM octopamine enhanced the amplitude of sensory-evoked EPSP of LG. Bath application of a cAMP analogue had a similar enhanced effect on LG. Under the adenylate cyclase inhibitor SQ22536, however, bath application of 5-HT caused no significant enhancement of the sensory-evoked EPSPs, although application of octopamine still increased the amplitude of the EPSP in LG. SQ22536 is an inhibitor of adenylate cyclase that catalyzes the synthesis of cAMP from ATP. These results strongly suggest that 5-HT-induced synaptic enhancement of the LGs was mediated by an increase in the cAMP level after activation of adenylate cyclase and that the effect of octopamine was cAMP-independent. In mammals, 5-HT receptors are grouped into seven classes (5-HT1–5-HT7) depending on their differences in response to pharmacology, structure, and transduction (for review, see Hoyer et al. 1994; Kroeze et al. 2002). Six classes are G-protein-coupled receptors and one class, 5-HT3 is a ligand gated ion channel. The mammalian 5-HT1 receptor is negatively linked to adenylate cyclase, whereas the 5-HT4 receptor in the hippocampus is associated with the activation of adenylate cyclase. Our study strongly suggests the existence of mammalian 5-HT4-like 5-HT receptor in the LG escape circuit in which activation elicits an increase in intracellular cAMP levels. At the moment, however, a receptor corresponding to the mammalian 5-HT4 class has not been cloned in invertebrates. The only putative mammalian 5-HT1 receptor that has been cloned is the 5-HT1crust receptor from some crustaceans, including crayfish (Sosa et al. 2004; Spitzer et al. 2005).
Site of action of 5-HT in the LG escape circuit
The sensory-evoked EPSP of both the early +' and later component increased in amplitude after bath application of 5-HT and the cAMP analogue, CPT-cAMP. A similar enhancement of LG excitability mediated by cAMP has also been reported by Edwards et al. (2002), although no actual responses of LGs recorded intracellularly were illustrated. In Aplysia, 5-HT facilitates the connections between siphon sensory neurons and motor neurons by increasing transmitter released from sensory neurons (Kandel 2001). 5-HT increases cAMP levels in a sensory neuron by activating adenylate cyclase. The cAMP, in turn, recruits the cAMP-dependent protein kinase A (PKA) in which activation causes phosphorylation of specific K+ channels. This results in a closure of K+ channels and a reduction of repolarizing K+ currents. The spike duration is prolonged and transmitter release from the sensory neurons is enhanced. Because sensory neurons innervating hairs on the surface of the tailfan make chemical synapses directly to the LGs and sensory interneurons (Araki and Nagayama 2003; Nagayama and Sato 1993), 5-HT might act on sensory afferents.
Some descending serotonergic neurons project into the terminal abdominal ganglion and appear to make direct contact with LG (Yeh et al. 1997). Our result that direct injection of the cAMP analogue, Sp-cAMPS into LG increased the peak amplitude of sensory-evoked EPSP suggests that 5-HT also acts on a transmembrane 5-HT receptor on LG. It still remains to be clarified how cAMP enhances the synaptic response or what type of cAMP-dependent protein kinase is recruited by cAMP in LG. The report that the postsynaptic injection of a Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, or EGTA into LGs inhibits long-term potentiation in LG and depression induced by tetanic sensory stimulation (Krasne and Edwards 2002) suggests a direct action of 5-HT on LG. Intracellular injection of Sp-cAMPS enhanced the early +' and later component of the EPSP. Because the ' component is mediated through monosynaptic chemical synapses from mechanosensory afferents and the component is mediated through electrical synapses from many intersegmental ascending interneurones (Araki and Nagayama 2003; Zucker 1972), cAMP must modulate not only chemical synapses but also electrical coupling of LGs. A report that suggests cAMP increases the conductance of gap junctions between CA3 pyramidal neurons in the rat hippocampus (Gladwell and Jefferys 2001) supports this idea. Further pharmacological and electrophysiological studies are needed to examine this possibility.
Octopamine-mediated second-messenger cascade
All of the octopamine receptors found so far in invertebrates belong to a G-protein-coupled superfamily, and most of them are coupled to activation of adenylate cyclase (Blenau and Baumann 2001; Roeder 1999), although several octopamine receptors in insects inactivate adenylate cyclase (von Nickisch-Rosenegk et al. 1996). The activation of a cAMP cascade by octopamine has been suggested to play a crucial role in learning and memory in insects (Hildebrandt and Müller 1995a,b; Müller 1997). In contrast to 5-HT application, the adenylate cyclase inhibitor SQ22536 did not inhibit the enhancing effect of octopamine on sensory-evoked EPSPs of LG. The synaptic responses of LG were increased by bath application of octopamine with and without precirculation of SQ22536. Furthermore, the rate of LG habituation was also similar under bath application of octopamine with and without precirculation of SQ22536, whereas the effect of 5-HT was suppressed by continuous perfusion of SQ22536. These results indicate that octopamine in LG escape system is not closely related to the activation of cAMP cascade, but that octopamine could stimulate certain intracellular messenger cascades exclusive of cAMP and cGMP. Some insect octopamine receptors are thought to be coupled to the IP3 system that activates the release of Ca2+ from internal stores (Baines and Downer 1994; Jahagirdar et al. 1987). Further physiological and pharmacological studies directed toward understanding the intracellular messenger cascades would provide a better understanding of the neural mechanism underlying crayfish escape.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Araki, Animal Behavior and Intelligence, Div. of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan (E-mail: arakim{at}sci.hokudai.ac.jp)
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REFERENCES |
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) increased after bath application of 5 µM 5-HT (Ai, top and middle). After washout, the response of LG recovered partially (Ai, bottom). To compare the peak amplitude of the EPSP response clearly, sweeps of control and 5 min after 5-HT application are superimposed (Aii). - - -, the onset of the disynaptically elicited 
, Ai). To compare the peak amplitude of the EPSP response clearly, sweeps of control and 5 min after octopamine application are superimposed (Aii). - - -, the onset of the disynaptically elicited 