Invert Neurosci (2015) 15:2 DOI 10.1007/s10158-015-0178-8 ORIGINAL PAPER Habituation of LG-mediated tailflip in the crayfish Toshiki Nagayama1 • Makoto Araki2 Received: 23 January 2015 / Accepted: 11 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015 Abstract Crayfish escape from threatening stimuli by tailflipping. If a stimulus is applied to the rear, crayfish escape up and forwards in a summersault maneuver that is mediated by the activation of lateral giant (LG) interneurons. The occurrence probability of LG-mediated tailflip, however, diminishes and habituates if a stimulus is repeatedly applied. Since crayfish have a relatively simple CNS with many identifiable neurons, crayfish represent a good animal to analyze the cellular basis of habituation. A reduction in the amplitude of the EPSP in the LGs, caused by direct chemical synaptic connection from sensory afferents by repetitive stimulations, is essential to bring about an inactivation of the LGs. The spike response of the LGs recovers within several minutes of habituation, but the LGs subsequently fail to spike when an additional stimulus is applied after specific periods following habituation. These results indicate that a decline in synaptic efficacy from the mechanosensory afferents recovers readily after a short delay, but then the excitability of the LGs themselves decreases. Furthermore, the processes underlying habituation are modulated depending on a social status. When two crayfish encounter each other, a winner–loser relationship is established. With a short interstimulus interval of 5 s, the rate of habituation of the LG in both socially dominant and subordinate crayfish becomes lower than in socially isolated animals. Serotonin and octopamine affect this social status-dependent modulation of habituation by means of & Toshiki Nagayama [email protected] 1 Department of Biology, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan 2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan activation of downstream second messenger system of cAMP and IP3 cascades, respectively. Keywords Chemical synapse Modulation Biogenic amines Second messengers Background Habituation is a well known form of non-associative learning (Thompson and Spencer 1966) in which reflexive behavioral responses gradually reduce upon repeated stimulation. Habituation is subject to plasticity in invertebrates, as has been shown in the siphon withdrawal reflex in Aplysia (Kandel 2001, 2009), the proboscis extension response of honeybees (Braun and Bicker 1992; Hammer and Menzel 1998) and the lateral giant (LG)-mediated tailflip of crayfish (Krasne 1969; Krasne and Woodsmall 1969; Zucker 1972b; Wine et al. 1975; Bryan and Krasne 1977; Fricke 1984; Marchand and Barnes 1992; Edwards et al. 1994; Krasne and Teshiba 1995; Edwards et al. 1999; Araki and Nagayama 2003; Edwards 2009; Nagayama and Newland 2011). Since the LG-mediated tailflip is a stereotyped behavior (Wiersma 1947) and crayfish have a relatively simple central nervous system with relatively few and often identifiable neurons (Wine 1984; Nagayama et al. 1993a, b, 1994), crayfish represent a good animal to analyze the cellular basis of habituation. If strong tactile stimuli are applied to the abdomen or tailfan, crayfish produce a rapid flexion of the abdominal musculature that leads to an escape response directed up and forwards in a summersault maneuver (Fig. 1a; also Wine and Krasne 1972). The lateral giant interneuron (LG; Fig. 1b) with a large diameter of ascending axon (Fig. 1c) acts as a command neuron by receiving sensory inputs directly and 123 2 Page 2 of 12 Invert Neurosci (2015) 15:2 Fig. 1 LG-mediated tailflip of the crayfish. a Tracing of LG tailflip. b Intracellular staining of the LG in the terminal abdominal ganglion. c Transverse section of abdominal third–fourth connective. These illustrations were modified from Nagayama (2008) indirectly from extero- and proprioceptive afferents (Wine and Krasne 1972; Newland et al. 1997; Araki and Nagayama 2003) and by making excitatory outputs with motor giant (MoG) motor neurons in anterior abdominal segments (Wine 1984). The rapid flexion of the abdomen is triggered within 10 ms following spikes in LG. The probability of occurrence of LG-mediated tailflip diminishes and habituates if a tapping stimulus to tailfan is repeatedly applied. Krasne and his colleagues show that tonic descending inhibition from higher center of brain via GABAergic pathway plays a role in controlling LG habituation that LGs show subthreshold response to stimulus (Krasne and Bryan 1973; Krasne and Wine 1975; Vu and Krasne 1992, 1993; Vu et al. 1993; Krasne and Teshiba 1995; Shirinyan et al. 2006). LGs also show habituation without descending inputs by severance of the nerve cord between thorax and abdomen or isolation of abdominal ganglia from the rest of body (Krasne 1969; Zucker 1972a, b; Wine et al. 1975). Krasne and Teshiba (1995) show that removing the influence of higher center on LG circuit reduces the tendency of the LG threshold. Thus, descending inputs from higher center control and/or modulate the activity of local regulation within the terminal abdominal ganglion. When a constant stimulus that sets just above the initial LG threshold is applied, the differences between intact and abdominal isolated animals are small (Wine et al. 1975). Here, we will focus on the local regulation 123 underlying habituation of LG neurones (=LG habituation) within the terminal abdominal ganglion using isolated nerve cord preparation with the results from our own laboratory to describe the synaptic mechanism of LG habituation, recovery from habituation, and social status-dependent modulation of habituation. Crayfish LG-flips and habituation To prepare crayfish for electrophysiological analysis (Fig. 2a), the nerve chain from the second to sixth (terminal) abdominal ganglia with relevant nerve roots is isolated from the rest of the body and pinned, dorsal side up, in a Sylgard-lined perfusion chamber, containing cooled physiological solution (van Harreveld 1936). The chamber is constantly perfused with fresh saline, and the bathing solution can be changed with a saline containing a specific drug. The dorsal ganglionic sheath of the terminal ganglion is surgically removed with fine forceps to facilitate the penetration of intracellular electrode and drug perfusion. The spike activity of LG is monitored extracellularly from the third–fourth abdominal connective using a suction electrode. Nerve roots 2, 3 and 4 of the terminal abdominal ganglion that contain the mechanosensory afferents innervating the uropods and telson are electrically stimulated simultaneously using a Invert Neurosci (2015) 15:2 Page 3 of 12 2 Fig. 2 LG habituation. a Experimental setup. b Responses of the LG to repeated sensory stimulation. When the stimulus is repeated with a 5-s interstimulus interval, the LG elicits spikes to the first three stimuli. From the fourth stimulus, the LG shows subthreshold responses. c Habituation curves. LG firing probability is plotted as the percentage of animals in which LG fired on a given trial single oil hook electrode, since the spike threshold of the LGs is relatively high. Square stimulus pulses (0.01–0.05 ms duration; 1–20 V intensity) are delivered through the stimulating electrode. Intracellular recordings from the LG, or relevant ascending interneurons, are made from their dendritic branches in the left half of the terminal ganglion neuropil with glass microelectrodes. After 15 min of rest following dissection, an intracellular microelectrode is driven into the neuropil of the terminal ganglion. The spike threshold of LG is then determined by applying a gradual increase in the intensity of stimulation of the sensory nerves. When LG produces an action potential, an extracellular spike with very large amplitude is recorded following the intracellular LG spike from the A3–A4 abdominal connective (Fig. 2b). After the LG spike threshold is determined, the intensity of stimulation is set so that the stimulus is just suprathreshold. The preparation is rested for a further 5 min before repeated sensory stimulation with the intended interstimulus interval. We have judged LG habituation to have occurred when LG failed to give rise to spikes following five continuous stimulus trials. In the recordings shown in Fig. 2b, the LG habituates from the fourth trial of the stimulus. In some preparations, the stimulus intensity is set just below suprathreshold to elicit LG spikes in order to analyze the synaptic response of the LGs more quantitatively. As sensory stimulation was repeated, the rate of firing probability of LGs in the tested populations declined depending on the stimulus intervals. When the stimulus is repeated with a 1-s interstimulus interval, the LG response immediately shows a rapid habituation, decreasing by 80 % of tested animals within four trials of stimulation, and by 95 % after the twentieth trial (filled circles in Fig. 2c). As the interstimulus interval is increased, the decrease in the LG response becomes slower. For example, at a 5-s interstimulus interval, the response of LG declines by 80 % after 20 trials (open circles in Fig. 2c) and at interstimulus intervals of 20 or 60 s, the response of LG decreases by 123 2 Page 4 of 12 50 % after 20 stimuli (Fig. 2c: filled triangles for 20-s intervals and open triangles for 60-s intervals). When a stimulus is repeated with a 300-s interstimulus interval, however, habituation occurs more readily (filled squares in Fig. 2c). The response of LG declines by 80 % after only 10 trials, and the decline of habituation curve is similar to that at a 5-s interstimulus interval. Thus, the number of stimuli necessary to cause habituation increases when the interstimulus interval is increased, with the exception of a 300-s interstimulus interval (Araki and Nagayama 2005). For example, the number of stimuli needed to cause habituation at a 5-s interstimulus interval is significantly greater than at a 1-s interstimulus interval (p \ 0.001; logrank test). Furthermore, with 20- and 60-s interstimulus intervals, the number of stimuli significantly increases compared to that with a 5-s interval (p \ 0.005 and p \ 0.001, respectively; log-rank test). The reason why the LGs habituated rapidly in response to stimuli at a 300-s interstimulus interval will be discussed later in the section referring to change in LG excitability. Synaptic mechanism of habituation The LGs receive sensory inputs through sensory neurons via direct synapses and by ascending interneurons via indirect synapses. Low intensities of sensory stimulation (=9 V, 10-ls duration) evoke compound potentials in the LG with two components (Fig. 3a top trace). As Krasne (1969) reported, the first EPSP, termed the a component, and a later EPSP, the b component, can be temporally discriminated. With further increases in stimulus intensity (=10 V), the stimulation evokes a spike rising from the beginning of b component (Fig. 3a 2nd trace). When the stimulus is repeated at the same intensity at 0.5 Hz, LG fails to give rise to a spike from the second stimulus (Fig. 3a third trace) and continues to respond with subthreshold EPSPs to later stimuli (Fig. 3a bottom trace). A comparison of the potentials elicited in LG evoked by stimulus pulses of 9-V intensity and 10-V intensity; the later part of a component increases following a spike from the b component (Fig. 3b top trace). A comparison of the potentials before and after LG habituation to the stimulus of 10-V intensity shows that the later part of the a component decreases in amplitude after habituation, while the amplitude of the early part of the a component remains constant (Fig. 3b middle trace). With repeated stimulation, the amplitude of the early part of the a component remains constant in amplitude, while that of the b component decreases gradually. The superimposed potentials of LG evoked in response to a subthreshold stimulus (9 V in intensity), and the response to the second stimulus at 10 V shows that the time course and amplitude of the a 123 Invert Neurosci (2015) 15:2 component vary little (Fig. 3b bottom trace). These results suggest that the potentials in LG could consist of three components when LG responds with a spike to sensory stimulation. The third component of EPSPs, termed as a0 component, is distinguishable at the boundary between the late a and early b component when the LG produces an action potential, but is difficult to discriminate after LG habituation (Araki and Nagayama 2003). Interneurons A and C, also known as NE-1 and RC-8 (Nagayama et al. 1993a), contribute to the b component of the LG response to sensory stimulation (Kennedy and Takeda 1965; Zucker et al. 1971; Zucker 1972a, b). Interneuron C responds to sensory stimulation with a train of spikes superimposed on a sustained membrane depolarization at stimulus intensities just suprathreshold for LG spikes (Fig. 3c left). When sensory stimulation is repeated, LG fails to give rise to a spike, which is indicated by the disappearance of the largest extracellular spike recorded from the third–fourth abdominal nerve cord (lower trace in Fig. 3c right). The spikes of interneuron C reduce in number following repeated stimulation (upper trace in Fig. 3c right), but the time course and amplitude of the initial spike of the interneuron do not change significantly before and after LG habituation. Since interneuron A has the next largest axon diameter to the giant interneurons (LG and MG), spikes of interneuron A are readily distinguishable by means of extracellular recording from the abdominal nerve cord (arrowheads in Fig. 3c lower trace). Interneuron A elicits spikes with rather constant latencies before and after LG habituation. Thus, these interneurons only show a slight reduction of activity in response to repeated stimulation, even though LG fails to give rise to a spike upon repeated sensory stimulation (Araki and Nagayama 2003). If EPSPs are mediated through chemical synapses, they are reduced in amplitude by the passage of depolarizing current and increased by hyperpolarizing current. By contrast, current injection usually has little effect on the size of potentials if they are mediated by electrical transmission (e.g., Nagayama et al. 1997b; Newland et al. 1997). Stimulation with an intensity set just subthreshold to elicit LG spikes evokes compound potentials in LG (upper trace in Fig. 4a). The passage of 1 nA hyperpolarizing current injected into LG causes an increase in amplitude of the sensory-evoked potentials. Superimposed sweeps to compare the EPSPs in LG at resting potential, and during hyperpolarizing current injection, show that the EPSP in the later part of the a component, that is the a0 component (asterisks in Fig. 4a), is increased in amplitude, while that of the early a component or the b component shows no change in amplitude (Fig. 4a). Since the a and b components of EPSPs are mediated through electrical synapses from sensory afferents and specific ascending interneurons, Invert Neurosci (2015) 15:2 Page 5 of 12 2 Fig. 3 Synaptic response of LG and interneuron C to sensory stimulation. a Subthreshold (top trace) and suprathreshold (second– fourth traces) stimuli. LG shows habituation from the second suprathreshold stimulus. Interstimulus interval is 1 s. b Superimposed sweeps of a. The a0 component of the EPSP is only observed when the LG gives rise to a spike. c Response of interneuron C before and after LG habituation. Arrowheads in bottom trace are extracellular spikes of interneuron A respectively (Zucker 1972a), this study suggests that LG receives chemically mediated inputs that are consistent with the a0 component directly from mechanosensory afferents innervating hairs on the surface of the tailfan (Araki and Nagayama 2003). Since mechanosensory afferents are known to release acetylcholine as an excitatory neurotransmitter (Miller et al. 1992; Ushizawa et al. 1996), the change in response of the LGs to sensory stimulation under bath application of d-tubocurarine, a nicotinic antagonist, is analyzed (Fig. 4b). The compound potentials in LG elicited by subthreshold stimulation decrease in amplitude about 6 min after bath application of 50 lL d-tubocurarine. The decrement in the amplitude of LG continues gradually (after 13 and 19 min) and recovers partially after about 30 min of washing with normal saline. This decrement in potentials could be resolved into two temporal phases. The later part of the a component, that is the a0 component, quickly reduces in amplitude (shown with asterisk in Fig. 4b), while the b component decreases gradually. This different time course of effects on the compound potentials suggests that two distinct chemically mediated inputs contribute to the compound potentials in LG. (Araki and Nagayama 2003). We further analyze the responses of LG to exteroceptive mechanical stimulation. Small numbers of mechanosensory 123 2 Page 6 of 12 123 Invert Neurosci (2015) 15:2 Invert Neurosci (2015) 15:2 b Fig. 4 Direct chemically mediated inputs from sensory afferents. a Injection of 1 nA hyperpolarizing current into LG increases the amplitude of the EPSP in the later part of a component, that is a0 component. Upper superimposed sweeps of the response of the LG are adjusted to the base level of each record. Lower superimposed sweeps are adjusted to the peak level of the EPSP. Asterisks indicate EPSP in a0 component The EPSP amplitudes of both the early a component and the b component are constant after injection of hyperpolarizing current. b Effect of bath application of d-tubocurarine on the response of LG to sensory stimulation. Superimposed sweeps of the LG response show that the a0 component of the EPSP (asterisk) disappears quickly and that of the b component diminishes gradually in amplitude. c Response of LG to hair stimulation. Superimposed sweeps triggered from each of two different afferents (c1 and c2) show that EPSPs consistently follow the sensory spike with a constant short latency. Superimposed sweeps triggered from the third afferent spikes (c3) show that the EPSP followed consistently with a constant long latency, and this EPSP decreased in amplitude under bath application of d-tubocurarine. d Schematic diagram of circuitry for LG tailflip hairs are deflected locally, and the response of LG was analyzed (Fig. 4c). Superimposed sweeps triggered from the spikes of a single afferent show that EPSPs in LG follow consistently with a constant short latency (Fig. 4c1). The EPSPs are about 0.9 mV in amplitude, have a rise time of about 0.8 ms, and a decay time to half amplitude of about 0.9 ms. There is a delay of about 0.8 ms between the afferent spike recorded in nerve root 3 and the start of the EPSP in LG. A second sensory afferent with a smaller amplitude spike in the extracellular recording is sampled sequentially (Fig. 4c2), and superimposed sweeps triggered from the spikes of this afferent show that EPSPs in LG follow consistently with a short constant latency of about 0.9 ms. The EPSPs are about 0.7 mV in amplitude and have a fast rise time of about 0.8 ms and a decay time to half amplitude of about 1.0 ms. Superimposed sweeps triggered from the spikes of the third afferent show that EPSPs in LG follow consistently (left trace in Fig. 4c3). There is, however, a rather long delay of about 1.5 ms between the afferent spike and the start of the EPSP in the LG. The EPSPs are about 0.8 mV in amplitude and have a rise time of about 2.4 ms. The falling phase declines gradually having a decay time to half amplitude of about 3.4 ms. They are, furthermore, decreased in amplitude under bath application of d-tubocurarine (right trace in Fig. 4c3). These results strongly indicate that the first two afferents make convergent electrical connections with the LG, while the third afferent makes chemically mediated synaptic transmission. Thus, LGs receive direct excitatory inputs from the mechanosensory afferents mediated through both electrical (that is a component) and chemical synapses (that is a0 component) with indirect electrical input via sensory interneurons (that is b component) as shown in Fig. 4d. The decrease in synaptic efficacy of the direct chemical synapses contributes, at least in part, to Page 7 of 12 2 elicit LG spikes and LG habituation (Araki and Nagayama 2003). Recovery from habituation then change in LG excitability The spike response of the LGs usually recovers quickly within minutes after habituation, but the LGs become less excitable when additional sensory stimuli are applied after a longer period following habituation (Araki and Nagayama 2005). For example, following a 1-min pause from habituation, additional sensory stimulation (=test stimulus) with the same intensity again elicits spikes in LG. By contrast, in animals rested for 30 min following habituation with no additional stimulation, the test stimulus fails to elicit a LG spike (Araki and Nagayama 2005). These observations suggest that habituation is caused by a decline in transmitter release from mechanosensory afferents (Zucker 1972b), but the synaptic efficacy of these sensory afferents could recover readily after a short delay that is sufficient to elicit a LG spike. Following recovery of the LG response, however, LG soon exhibits a reduction in excitability for spike generation after certain periods of delay. The a0 component of the EPSP disappears after habituation, while the a0 component of the EPSP is observed when the test stimulus is applied (Araki personal observation). At the moment, the neural mechanisms underlying this decrease in excitability of LGs remain unclear, but the observation that the threshold of LG following the change in LG excitability becomes significantly higher than the threshold just after habituation (Araki and Nagayama 2005) suggests some physiological change could occur in the LGs during the period of the delay following habituation. As the interstimulus interval becomes shorter, the LG habituation occurs rapidly, but a longer delay is necessary to decrease excitability of LGs. As the interstimulus interval is increased, the delay needed for decrease in excitability becomes shorter (Araki and Nagayama 2005). The observation that LG habituates rapidly to repetitive stimulation with a 300-s interstimulus interval (Fig. 2c) would explain that the LG response shifts directly from habituation to reduction of excitability during the course of longer periods of stimulation. Status-dependent modulation of habituation Animal behaviors are modulated according to external and internal conditions. For example, the crayfish LG response habituates more slowly when animals are exposed in high temperatures (Nagayama and Newland 2011). Furthermore, the establishment of social hierarchies also affects 123 2 Page 8 of 12 Invert Neurosci (2015) 15:2 crayfish behaviors. For example, a gentle mechanical stimulation to the tailfan evokes an avoidance reaction when a crayfish shows a stationary resting posture (Nagayama et al. 1986). Small crayfish show an escape-like dart response, while larger animals show a defensive-like turn response. When two small crayfish encounter each other, a winner–loser relationship is established following several combats, with the winner changing its response to show a turn response (Fujimoto et al. 2011). The establishment of social status also affects excitability of LGs (Krasne et al. 1997). Subordinate animals exhibit an increased threshold for LG activation. LG habituation is also modulated by state-dependent manner (Araki et al. 2013). When a sensory stimulus is applied repeatedly with a 5-s interstimulus interval, LG from socially isolated crayfish rapidly habituates, with a decrease in firing probability by 50 % within four trials of stimulation, and by 75 % after 25 trials (Fig. 5a, filled triangles). By contrast, LG’s responses from socially subordinate crayfish show a slower rate of habituation (Fig. 5a, open circles). After the twentieth trial of stimulation, only 50 % of subordinate animals show LG habituation, while 35 % still respond with a spike after 40 trials. Dominant crayfish are also found to show a slow decline in the rate of LG habituation in the isolated abdominal nerve cord (Fig. 5a, filled circles). Approximately 20 trials are needed to decrease the LG firing probability by 50 %, while more than 40 % still give rise to a spike after 40 trials. The stimulus number required to habituate LG in both the dominant and subordinate animals increases significantly in comparison with socially isolated crayfish (p \ 0.01; log-rank test). This status-dependent change in LG habituation is maintained for at least a week (Fig. 5b). The decline in the habituation curve is very similar for dominant animals of both first day and seventh day. On the seventh day, about 40 % of animals do not show LG habituation within 40 trials of stimulation. Fig. 5 Status-dependent modulation and the effect of biogenic amines on habituation. a Habituation curves of the response of LG to repeated sensory stimulation with a 5-s interstimulus interval. The LG firing probabilities of control (filled triangles), dominant (filled circles), and subordinate (open circles) animals are plotted. b Longterm memory of status-dependent modulation of habituation. Habituation curve of the response of LG in dominant crayfish on the seventh day to repeated sensory stimulation with a 5-s interstimulus interval. c Habituation curves of the response of LG to repeated sensory stimulation with a 5-s interstimulus interval under bath application of biogenic amines. The LG firing probabilities of control (gray squares), 5 lM serotonin (open circles) application and 10 lM octopamine (filled circles) application are plotted. d Effect of serotonin (d1) and octopamine (d2) on synaptic response of the LG to the sensory stimulation. Bath application of 5 lM serotonin (d1) or 10 lM octopamine (d2) enhances both the a ? a0 components and the b component of the EPSPs. Asterisk indicates spike of LG 123 Invert Neurosci (2015) 15:2 Since subordinate animals show mainly submissive acts such as retreats and tailflips in response to the attacks of dominant animals (Sato and Nagayama 2012; Ueno and Nagayama 2012), it would be reasonable that subordinate animals show a slow decline in spike activity of the LGs to repeated sensory stimulation, since a decrease in the rate of habituation would be necessary to evade repeated attacks of dominant animals. By contrast, the result that the rate of habituation of dominant crayfish is also less than control animals is contradictory since dominant animals perform more aggressively and appeared not to need to evade encounters from subordinates. Herberholz et al. (2001) have reported that crayfish frequently show offensive tailflips during agonistic encounters. Offensive tailflip begins with an abdominal extension followed by abdominal flexions and re-extensions. The abdominal extensions are accompanied by a spread of the tailfan that is maintained during the abdominal flexion, which occurred primarily around the anterior abdominal segmental joints, while the posterior segments remained extended. This configuration helps to throw the animal up into the water column above the opponent and could act to allow a crayfish to abruptly change orientation in a short period, i.e., during the LG-mediated tailflip. Offensive tailflip occurs with a short interval of less than 5 s before the dominance order is determined. Dominant animals show a decrease in the rate of habituation in response to sensory stimulation with a 5-s interstimulus interval. It would be advantageous therefore to prevent habituation of tailflip with short stimulus intervals during agonistic bouts. Sensory reception between LG and offensive tailflip might be linked causally, and a common neural mechanism could occur to prevent habituation. In fact, with a long interstimulus interval of 60 s, the rate of habituation of dominant animals is similar to that of socially isolated animals, although subordinate animals still show a slow rate of the habituation (Araki et al. 2013). The neuromodulators, serotonin, and octopamine play a key role in dominance hierarchy formation (Huber et al. 1997; Huber and Delago 1998). Direct injection of serotonin or octopamine into the systemic circulation of crayfish induces dominant-like or subordinate-like status and motivation, respectively (Momohara et al. 2013). Serotonin and octopamine also affect the responsiveness of LG to sensory stimulation (Glanzman and Krasne 1983; Yeh et al. 1997; Edwards et al. 2002; Krasne and Edwards 2002; Antonsen and Edwards 2007; Lee et al. 2008) and increase the number of stimuli required to habituate the LG response to sensory stimulation with a 5-s interstimulus interval (Araki et al. 2005). Under bath application of 5 lM serotonin (open circles in Fig. 5c) and 10 lM octopamine (filled circles in Fig. 5c), the rate of decease in the response of LG to sensory stimulation is slower. Only 20–35 % of animals fail to respond with a spike within 5 trials of Page 9 of 12 2 stimulation. The response of LG decreases by 50 % just after 15 stimuli, and approximately 30 % of animals still respond with a spike after 40 stimuli. Thus, both serotonin and octopamine decrease the rate of habituation, and the numbers of stimuli needed to habituate LG increase significantly (p \ 0.05; log-rank test), as is the case of both dominant and subordinate animals. The sensory-evoked EPSP of LG is significantly increased in amplitude, including both the early a ? a0 component and the later b component under bath application of 5 lM serotonin (Fig. 5d1) or 10 lM octopamine (Fig. 5d2). If the stimulus intensity of sensory stimulation is set subthreshold for LG spikes during serotonin or octopamine application, sensory-evoked EPSPs of the LGs frequently induce spikes as shown in Fig. 5d2. These findings suggest that the increment in the number of stimuli required to habituate the LG response to sensory stimulation is possibly linked to serotonin and octopamine levels of dominant and subordinate crayfish. Serotonin and octopamine enhance the LG responsiveness to sensory stimulation (Araki et al. 2005; Araki and Nagayama 2012). The majority of serotonin and octopamine receptors belong to a superfamily of G-protein-coupled receptors, and their effects are mediated by second messengers (Hoyer et al. 1994; Gerhardt et al. 1997; Roeder 1999). We therefore confirmed the effects of second messengers upon LG response to the sensory stimulation. When a cAMP analogue, sp-cAMPS, is iontophoretically injected into LG, the amplitude of EPSPs in LG to sensory stimulation is increased significantly (Fig. 6a). Similarly, intracellular injection of IP3 agonist, adenophostin A, into the LG also enhances the LG response to stimulation (Fig. 6b). Bath application of a cGMP analogue has no obvious effect upon the process of LG habituation (Araki et al. 2005). Following activation of adenylate cyclase, cAMP level is increased, while the level of IP3 increases following activation of phospholipase C. The enhanced effect of bath application of serotonin (Fig. 5d1) is not detected under bath application of the mixture of serotonin and adenylate cyclase inhibitor, SQ22536 (Fig. 6c1). On the other hand, an enhancing effect of octopamine (Fig. 5d2) is unchanged under SQ22536 (Fig. 6c2). Thus, the effect of serotonin appears to be mediated by an increase in the level of cAMP. Intracellular injection of U-73122, a phospholipase C inhibitor, into the LG has no obvious effect on the effect of serotonin on the LG response to sensory stimulation (Fig. 6d1). Enhancement of LG response to sensory stimulation, especially that of early a and a0 components mediated by octopamine, is canceled by intracellular injection of U-73122 into LG (Fig. 6d2). b component of the EPSP is not affected significantly since responses of sensory interneurons to sensory stimulation also increase during bath application of octopamine. Thus, effect of 123 2 Page 10 of 12 Invert Neurosci (2015) 15:2 Fig. 6 Modulatory effects of second messengers on LG response to sensory stimulation. a Iontophoretically injected 50 lM Sp-cAMPS into LG enhances the sensory-evoked EPSP of both the a ? a0 components and the b component. b Iontophoretic injection of 100 lM adenophostin A into LG enhances the sensory-evoked EPSP of both the a ? a0 components and the b component. c Effect of bath application of the adenylate cyclase inhibitor SQ22536 of 100 lM in concentration upon the enhancing effect of serotonin (c1) and octopamine (c2). The serotonin-induced enhancement of the sensory-evoked EPSP in LG is canceled, while the octopamineinduced enhancement is not affected. d Effect of iontophoretic injection of the phospholipase C inhibitor U-73122 of 40 lM in concentration into the LG upon the enhancing effect of serotonin (d1) and octopamine (d2). Serotonin-induced enhancement of the sensoryevoked EPSP of the LG is not affected, but the octopamine-induced enhancement of sensory-evoked EPSP of the a ? a0 components is canceled. Asterisks indicates spikes of LG octopamine is mediated by an increase in the level of IP3 (Araki and Nagayama 2012). The second messenger, cAMP system, is linked to the serotonin-induced synaptic enhancement of the LG response, while IP3 system is linked to the octopamine-induced synaptic enhancement of LG. physiologically and morphologically (Wine 1984; Nagayama et al. 1993a, b, 1994), their neurotransmitters have been characterized (Ushizawa et al. 1996; Nagayama et al. 1997a, 2004; Aonuma and Nagayama 1999), and the pharmacological profiles of receptors have been clarified (Miyata et al. 1997; Nagayama 2005; Sosa et al. 2004; Spitzer et al. 2005, 2008). Thus, the cellular mechanisms for habituation are accessible behaviorally and neurophysiologically. Furthermore, the process of habituation shows status-dependent plasticity depending on the effects of biologic amines, e.g., serotonin and octopamine. Habituation of the LG escape reaction is known to be retained for several hours in intact animals (Krasne and Woodsmall 1969; Wine et al. 1975). Furthermore, dominant animals Conclusion The LG tailflip of the crayfish is a good system to elucidate the neural basis of habituation, since the LG tailflip is a stereotyped behavioral act and that many neurons contributing to the LG system are identifiable both 123 Invert Neurosci (2015) 15:2 show a slow rate of habituation a week after they became dominants. Descending tonic inputs are thought necessary to maintain habituation for long periods (Krasne and Teshiba 1995). The action of serotonin and octopamine is related to the activation of cAMP and IP3s messenger systems (Araki et al. 2005; Araki and Nagayama 2012). Since descending inputs from brain exert moment-to-moment control over LG’s excitability and habituation (Krasne and Teshiba 1995; Shirinyan et al. 2006), further studies to clarify the interactions between descending inputs and the local regulation of LG habituation within the terminal abdominal ganglion will provide the neurophysiological basis for the long-term memory of habituation. The neural circuitry that produces the LG tailflip is similar to that underlying the fast startle response, the C-start, of teleost fishes. Mauthner neurons in the hindbrain have descending large diameter axons that are responsible for triggering this response (Eaton et al. 1981). Like crayfish LGs, the Mauthner neurons also receive sensory inputs directly from sensory afferents via electrical synapses and indirectly from sensory interneurons via chemical synapses. Although the Mauthner neuron is not command neuron, it belongs to a class of neurons called reticulospinal neurons. The C-start response is also known to show habituation (Roberts et al. 2011). To compare the homology of neural mechanisms for habituation of both animals would be interesting to better understand the convergent evolution of neural circuitry. Acknowledgments This work was supported by grants from the Ministry of Education, Science, Sport, Culture and Technology to T.N. We are grateful to Dr. H. Aonuma for his assistance of this work. Conflict of interest None. References Antonsen BL, Edwards DH (2007) Mechanisms of serotonergic facilitation of a command neuron. J Neurophysiol 98:3494–3504 Aonuma H, Nagayama T (1999) GABAergic and non-GABAergic spiking interneurons of local and intersegmental groups in the crayfish terminal abdominal ganglion. J Comp Neurol 410:677–688 Araki M, Nagayama T (2003) Direct chemically mediated synaptic transmission from mechanosensory afferents contributes to habituation of crayfish lateral giant escape reaction. 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