Sensory Activation and Receptive Field Organization of the Lateral Giant Escape in Crayfish Yen-Chyi Liu and Jens Herberholz J Neurophysiol 104:675-684, 2010. First published 26 May 2010; doi:10.1152/jn.00391.2010

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Journal of Neurophysiology publishes original articles on the function of the . It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2010 by the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at http://www.the-aps.org/. J Neurophysiol 104: 675–684, 2010. First published May 26, 2010; doi:10.1152/jn.00391.2010.

Sensory Activation and Receptive Field Organization of the Lateral Giant Escape Neurons in Crayfish

Yen-Chyi Liu1 and Jens Herberholz1,2 1Department of Psychology, 2Neuroscience and Cognitive Science Program, University of Maryland, College Park, Maryland

Submitted 28 April 2010; accepted in final form 26 May 2010

Liu YC, Herberholz J. Sensory activation and receptive field 1999; Herberholz 2007; Krasne and Edwards 2002a; Wine and organization of the lateral giant escape neurons in crayfish. J Krasne 1982). Moreover, escape circuit activation can be Neurophysiol 104: 675–684, 2010. First published May 26, 2010; recorded in freely behaving allowing for a noninvasive doi:10.1152/jn.00391.2010. Crayfish (Procambarus clarkii) have bilateral pairs of giant that control rapid escape measure of neural activity patterns under more natural condi- movements in response to predatory threats. The medial giant tions (Herberholz 2009; Herberholz et al. 2001, 2004; Liden neurons (MGs) can be made to fire an by visual or and Herberholz 2008). The crayfish escape systems also pro- tactile stimuli directed to the front of the and this leads to vide a rare opportunity to test the effects of neuromodulators an escape tail-flip that thrusts the animal directly backward. The on identified neurons and small neural networks (Antonsen and Downloaded from lateral giant neurons (LGs) can be made to fire an action potential Edwards 2007; Edwards et al. 2002; Teshiba et al. 2001; Yeh by strong tactile stimuli directed to the rear of the animal, and this et al. 1996). produces flexions of the abdomen that propel the crayfish upward and forward. These observations have led to the notion that the The fastest and most powerful escape responses in crayfish receptive fields of the giant neurons are locally restricted and do are generated by pairs of bilateral command neurons, the not overlap with each other. Using extra- and intracellular electro- lateral giant interneurons (LGs) and the medial giant interneu-

physiology in whole animal preparations of juvenile crayfish, we rons (MGs). More than 60 years ago, it was first discovered jn.physiology.org found that the receptive fields of the LGs are far more extensive that firing of any of the crayfish’s giant interneurons caused than previously assumed. The LGs receive excitatory inputs from stereotyped tail flexions and that the LGs could only be descending interneurons originating in the ; these interneu- recruited by stimulation of the posterior half of the body and rons can be activated by stimulation of the antenna II or the protocerebral tract. In our experiments, descending inputs alone the MGs only by stimulation of the anterior half (Wiersma could not cause action potentials in the LGs, but when paired with 1947, 1961). Since then, numerous behavioral and physiolog- excitatory postsynaptic potentials elicited by stimulation of tail ical studies have confirmed this relationship between on February 10, 2012 afferents, the inputs summed to yield firing. Thus the LG escape orientation and activation of the giant interneurons. Only neurons integrate sensory information received through both ros- tactile stimuli directed to the rear of the crayfish can fire the tral and caudal receptive fields, and excitatory inputs that are LGs and elicit an escape tail-flip that pitches the animal up- and activated rostrally can bring the LGs’ membrane potential closer to forward away from the stimulus. MG-mediated tail-flips, how- threshold. This enhances the animal’s sensitivity to an approaching ever, only happen in response to strong tactile or visual stimuli predator, a finding that may generalize to other species with directed to the front and propel the crayfish backward (Ed- similarly organized escape systems. wards et al. 1999; Liden and Herberholz 2008; Wine and Krasne 1972, 1982). In addition, attacks from natural predators INTRODUCTION evoke LG tail-flips when the attack is directed at the tail and abdomen or MG tail-flips when the attack is directed toward Juvenile crayfish typically inhabit the more shallow areas of the head and thorax (Herberholz et al. 2004). streams and ponds and are most commonly preyed on by fish, Of the two giant circuits, the LG circuit has received most wading birds, and mammals (Correia 2001; Davis and Huber experimental attention due to the accessibility of its individual 2007; Englund and Krupa 2000). The response to attacks from components, and it is now considered one of the best under- these predators must be prompt and appropriate to ensure stood neural circuits in the entire animal kingdom (Edwards et survival and future reproductive success (Lima and Dill 1990). al. 1999; Krasne and Edwards 2002a,b). Previously identified Studies that examined predation on crayfish in natural habitats excitatory inputs to the LGs are purely mechanosensory, and reveal little detail of the actual escape behavior but emphasize they are generated by primary afferents and sensory interneu- the ecological implications of predator-prey interactions (e.g., rons located in the abdomen and tail (Wine and Krasne 1972). Blake and Hart 1995; Garvey et al. 1994; Söderbäck 1994; The primary afferents are excited by movement of sensory Stein and Magnuson 1976). Studying escape behavior of cray- hairs and by mechanical displacement of tail segments (Ebina fish in the laboratory, however, has been a popular choice. In and Wiese 1984; Newland et al. 1997), and they are coupled to addition to stereotyped behaviors that can be repeatedly acti- each other to amplify the excitatory input to the LGs (Antonsen vated, the underlying neural circuits are characterized by large, et al. 2005; Herberholz et al. 2002). The afferents connect identifiable neurons that can be easily accessed in dissected directly to the LGs (Araki and Nagayama 2003; Zucker et al. preparations (Edwards and Herberholz 2005; Edwards et al. 1971) and to mechanosensory interneurons; if a critical number Address for reprint requests and other correspondence: J. Herberholz, Dept. of these interneurons is recruited, they cause an action potential of Psychology, and Cognitive Science Program, University of in the LGs (Zucker 1972; Zucker et al. 1971). The LGs then Maryland, College Park, MD 20742 (E-mail: [email protected]). make powerful electrical output connections to pairs of giant www.jn.org 0022-3077/10 Copyright © 2010 The American Physiological Society 675 676 Y.-C. LIU AND J. HERBERHOLZ motor neurons (MoGs), which innervate flexor muscles that The temperature inside the tubes was monitored using a thermometer, bend the abdominal segments (Mittenthal and Wine 1973). and animals were kept in the ice until the temperature in the tube had Together, these experiments further supported the notion cooled down to 1.5°C. All dissections and experiments were per- that the receptive fields of the giant interneurons are nonover- formed in buffered (pH 7.4) crayfish saline (in mM: 202 NaCl, 5.37 KCl, 13.53 CaCl , 2.6 MgCl , and 2.4 HEPES). Animals were firmly lapping and the LGs’ excitatory inputs are confined to the 2 2 pinned ventral side up in a Petri dish lined with silicone elastomer abdomen and tail. However, the possibility that the LGs re- (Sylgard), and the entire preparation was bathed in fresh, noncircu- ceive excitatory inputs that descend from rostral sensory sys- lating saline. The temperature in the dish stayed constant at 18–19°C tems was never excluded because most studies on the LG throughout the experiments. escape circuit were performed in isolated crayfish abdomens The exoskeleton directly above the ventral nerve cord was removed where any descending inputs were eliminated (e.g., Araki and so that all abdominal segments and primary tail afferents were Nagayama 2005; Krasne 1969; Roberts 1968; Yeh et al. 1997), exposed (Fig. 1A). 1–3 were cut in all ganglia except the last or the existence of excitatory inputs from the front was not abdominal ganglion (A6). The ventral nerve cord was flipped over and explicitly measured (Herberholz et al. 2004; Wine and Krasne pinned down by the nerves so that the dorsal side containing the LG 1972). Descending inputs to the LGs that originate in the neurons was facing up. We paid attention to keep the ventral artery rostral areas of the nervous system have been described; intact, although some damage may have occurred in some prepara- tions when the cord was flipped over. To expose the caudal area of the however, they are considered to be entirely inhibitory (Krasne brain and provide access to the initial segments of the brain connec- and Teshiba 1995; Krasne and Wine 1975; Vu and Krasne tives, the first, second, and third maxillipeds, a small part of exoskel- 1992; Vu et al. 1993). Here, we show for the first time that the eton located between the remaining mouthparts and the nephropores,

LGs receive subthreshold excitatory inputs from descending as well as both antennal glands, were removed (Fig. 1A). For stimu- Downloaded from interneurons that are activated by stimulation of rostrally lo- lation of rostral sensory systems, a small window was cut into the fifth cated sensory systems. These additional inputs bring the LG basal segment of the right antenna (posterior to the first segment of the closer to threshold and, when activated by an approaching flagellum) to expose the antenna II nerve or into the caudal part of the predator, could prepare the animal for an attack that is ulti- right eyestalk and the underlying sheath to expose the protocerebral mately directed toward its abdomen or tail. Parts of the results tract. Status of the animals was consistently monitored by observing have been published previously in abstract form (Herberholz spontaneous neural activity in the sensory nerves, brain connectives and the ventral nerve cord. Experiments were only conducted in and Liu 2008). preparations that appeared healthy and lively, which most remained jn.physiology.org for several hours after the dissection. METHODS Animals and dissecting procedure Electrophysiology Juvenile crayfish (Procambarus clarkii) of both sexes were ob- Schematics illustrating the respective positions of recording elec- tained from a commercial supplier and kept in communal tanks before trodes and stimulating electrodes for each experiment are shown in on February 10, 2012 the experiments. Animals were fed medium-sized shrimp pellets twice Figs. 2–5 for clarity. a week and kept under a constant 12 h:12 h light-dark cycle. Crayfish The basic organization of electrode placement was as follows (Fig. used in experiments (n ϭ 36; size: 3.7 – 4.3 cm; measured from 1B): two silver wire hook electrodes were placed on the ventral nerve rostrum to telson) were thoroughly checked for intactness, and those cord (VNC), one rostral and one caudal to the LG recording site to missing limbs or sensory appendages and those with soft exoskel- monitor evoked neural activity in the nerve cord. Silver wire hook etons, indicating a recent molt, were excluded. electrodes were also placed on the brain connectives (BC) for record- Minimal invasive surgery was used to provide local access to the ings of activity. For stimulation of sensory afferents and nervous system in largely intact whole animal preparations. The interneurons, hook electrodes were placed on the peripheral nerves of dissecting procedure was modified using a previously described tech- A6 to stimulate primary tail afferents and on the antenna II nerve in nique (Glantz and Viancour 1983; Herberholz and Edwards 2005; the fifth basal segment of the antenna or on the protocerebral tract just Herberholz and Liu 2008). Animals were anesthetized by placing posterior of the lateral protocerebrum. The antenna II nerves contain them into small plastic tubes and inserting the tubes into crushed ice. axons of primary afferents from and chemorecep-

A B Protocerebral tract FIG. 1. Minimal invasive surgery was performed to provide Head local access to the nervous system in largely intact whole- Brain animal preparations. A: part of the exoskeleton and the mouth- parts were removed to provide access to the brain and brain Antenna II Nerve connectives (BC). The ventral nerve cord (VNC) was exposed by removing tissue between the 1st and last abdominal ganglia. MG & INT For stimulation of rostral sensory systems, a small window was Brain connectives cut into the 5th basal segment (posterior to the flagellum) of the (BC) right 2nd antenna to expose the antenna II nerve, or into the A1 caudal part of the right eyestalk to expose the protocerebral A2 tract (not shown). B: wire hook electrodes were placed on the A3 ventral nerve cord and on the brain connectives for recordings LGs Abdominal of interneuronal activity. Hook electrodes were placed on the A4 ganglia peripheral nerves of A6 to stimulate primary tail afferents and on Ventral nerve cord A5 the antennal nerve or protocerebral tract to stimulate sensory (VNC) A6 afferents and/or interneurons. Intracellular microelectrodes were used to record and stimulate the lateral giant neurons (LGs) in the VNC of the abdomen, and the medial giant neurons (MGs) as well Abdomen as other interneurons of interest in the BC. A6 sensory nerves

J Neurophysiol • VOL 104 • AUGUST 2010 • www.jn.org ROSTRAL SENSORY EXCITATION OF THE LATERAL GIANT NEURONS 677 tors located on the flagellum, and axons of proprioreceptors in its Stimulating the antenna II nerve at low intensity caused basal segments, all of which project to the antenna II neuropils smaller depolarizations in MG and LG and the activation of a (Sandeman et al. 1992). The protocerebral tract contains axons of large single descending unit recorded in the BC and VNC (Fig. primary visual afferents, second order visual interneurons, multimodal 2B). By comparing the positions of the recorded action poten- sensory interneurons, all of which descend to the median protocere- tial in the BC and at different locations of the VNC, it became brum; in addition, the protocerebral tract also contains the axons of ascending visual interneurons from the contralateral eye, and—within apparent that the action potential originated in the brain region the olfactory globular tract—small diameter axons of ascending ol- and descended from head to tail. Weak stimulation (1.2 V, 0.3 factory interneurons which originate in the accessory and olfactory ms) produced no measurable activity in the BC or VNC and lobes (Mellon 2000; Sullivan and Beltz 2001). produced no visible depolarization of LG (gray traces). A Intracellular microelectrodes for recording and current injection slightly stronger stimulus (1.4 V, 0.3 ms) resulted in the had resistances of 15–35 M⍀ and were filled with 3 M potassium recruitment of an action potential that was seen in the BC with chloride (KCl) for most experiments. Because inhibitory postsynaptic a delay of 9.3 ms after the stimulus onset and arrived 3 ms later potentials in LG are mediated by GABA through an increase in at the rostral recording site of the VCN (between the 1st and chloride conductance (Roberts 1968; Vu et al. 1993), leak of chloride 2nd abdominal ganglia) and 1.1 ms later in the caudal record- into the impaled could potentially change an inhibitory ing site of the VCN (between the 4th and 5th abdominal postsynaptic potential (IPSP) into an excitatory PSP (EPSP). Because this may cause a problem for unambiguous identification of LG ganglia). The temporal pattern of spike propagation is consis- EPSPs in small animals like the ones we used in our study, we also tent with the possibility that this was in fact a single descending used microelectrodes filled with 2 M potassium acetate (KAc; 15–40 unit (Fig. 2B; arrows), which caused the depolarization (0.7 ⍀ M ) to conduct several control experiments. mV) in the LG (black traces). Using electrodes filled with 2 M Downloaded from LG neurons were impaled with one or two microelectrodes in close KAc (n ϭ 4) instead of 3 M KCl produced very similar results proximity and anterior to the fourth (A4) or sixth (A6) abdominal (Fig. 2B1). A weaker stimulus (3.6 V) applied to antenna II ganglion. MGs and other interneurons of interest were impaled with nerve had no measurable effect (gray traces), whereas a electrodes in the brain connectives close to the caudal part of the slightly stronger stimulus (4.0 V) led to recruitment of a single brain. Giant neurons were identified by their response to rostral or descending unit that elicited a unitary PSP (0.85 mV) in LG caudal sensory nerve stimulations; unambiguous identification was (black traces). also warranted by measurements of size and conduction velocity of giant neuron action potentials evoked with suprathreshold current Within each preparation, the temporal relationship between jn.physiology.org injections. stimulus onset, recorded interneuron spike and corresponding We used microelectrode amplifiers (Axoclamp900A; Molecular LG PSP was highly consistent for each stimulus intensity. Five Devices) for current clamp experiments, a Grass stimulator (Model separate stimuli (2.1 V, 0.3 ms) successively applied to the S88) and an A-M Systems differential amplifier (Model 1700) for antenna II nerve recruited the same interneuron and the corre- stimulation and recording through hook electrodes. Analog data were sponding LG PSP each time (Fig. 2C; 5 traces superimposed). digitized using a Digidata 1440A (Molecular Devices) and stored and The same result was observed when using KAc-filled micro- on February 10, 2012 analyzed using pClamp10.0 and Clampfit 10.0 software (Molecular electrodes (n ϭ 2) to record LG PSPs (Fig. 2C1). Five Devices). repetitive stimuli (4.0 V, 0.3 ms) to the antenna II nerve recruited the same interneuron and led to corresponding PSPs RESULTS in LG. ϳ LG receives inputs from rostrally located sensory systems Conduction times between stimulus site and LG ( 13 ms) were similar for most experiments (Fig. 2, B and C), suggesting To measure evoked neuronal activity and directionality of that the same interneuron may have been recruited in these action potential propagation, we placed hook electrodes on the cases. We only recorded inputs to the ipsilateral LG near A4 exposed antenna II nerve or protocerebral tract for stimulation, and A6; although we did not measure LG responses in A5 or on the ipsilateral BC, and on two separate locations on the in any of the ganglia that are located more anterior to A4, ventral nerve cord either anterior (VNC_rostral) or posterior results are not expected to be different. (VNC_caudal) to the LG intracellular recording site. One LG also received inputs from descending units activated by intracellular electrode was inserted into the MG neuron near its stimulation of the protocerebral tract (n ϭ 4, Fig. 3). By initial segment close to the caudal part of the brain, and a varying the intensity of the stimulus, an increasing number of second intracellular electrode was inserted into the LG ipsilat- descending units was activated. A weak stimulus (4.6 V, 0.3 eral to the respective stimulation site (i.e., antenna II nerve or ms) produced one measurable action potential in the VNC and protocerebral tract). Recordings from the LG in abdominal produced only a small depolarization of LG (0.5 mV; light gray segments A4 (n ϭ 22) and A6 (n ϭ 8) of different animals traces in Fig. 3A). A second interneuron was recruited with were obtained with similar results using KCl- or KAc-filled stronger electrical stimulation (5 V, 0.3 ms) and was found electrodes. propagating through the BC (not shown) and VNC, resulting in Single-pulse electrical stimulation of the right antenna II a larger depolarization of LG (1.1 mV; dark gray traces in Fig. nerve caused depolarizations in both the ipsilateral MG and the 3A). Higher stimulus intensity (5.4 V, 0.3 ms) resulted in ipsilateral LG (Fig. 2). Electrical stimulation of this nerve also activation of more descending units, producing a larger, com- produced action potentials in interneurons, which are recorded pound PSP in LG (1.7 mV; black traces in Fig. 3A). The in the right BC and in both locations of the VNC. With experiment was repeated twice with KAc solution in the increasing stimulus intensity (Fig. 2A; gray traces: 3 V, black electrode and produced similar results (Fig. 3A1). Gradual traces: 3.5 V), an increasing number of interneurons was increases in stimulus voltage (light gray traces: 2 V, dark gray recruited and corresponding changes of the MG PSP and the traces: 3 V, black traces: 4 V) applied to the protocerebral tract LG PSP were seen (Fig. 2A). led to the activation of additional descending interneurons and

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FIG. 2. Single-pulse electrical stimulation of the right antenna II nerve causes depolarizations in the ipsilateral MG and LG. A: electrical stimulation of the antenna II nerve produces action potentials in activated neurons, which can be seen in the right BC and in both recording sites of the VNC (gray traces). With increasing stimulus intensity (black traces), an increasing number of interneurons are recruited and corresponding changes of the postsynaptic potentials in MG and LG can be seen. B: stimulating the antenna II nerve at low intensity causes smaller depolarizations in MG and LG and the activation of a large single descending unit recorded in the BC and VNC. Weak stimulation produced no measurable activity in the BC or VNC and produced no visible depolarization of LG (gray traces). A slightly stronger stimulus resulted in the recruitment of a descending action potential that depolarizes LG (black traces). B1: the results obtained with KCl-filled electrodes were replicated in different animals using electrodes filled with KAc. C: 5 superimposed traces of the BC and LG recordings after 5 separate stimuli (same voltage) were applied to the antenna II nerve showing that the recorded action potentials and LG PSPs are time-locked to the stimulus. C1: the results obtained with KCl-filled electrodes were replicated in different animals using electrodes filled with KAc. produced an increase in both the number and size of the LG sensory inputs were combined with sensory inputs from pri- PSPs. mary afferents of the tail. Descending inputs in these experi- ments could not alone cause action potentials in the LGs, but Combined sensory input to LG can elicit an action potential when paired with EPSPs elicited by stimulation of tail affer- ents, the inputs summed to yield firing (Fig. 4A). Hook elec- To verify the excitatory nature of the descending inputs to trodes were placed on the antenna II nerve and on sensory LG, two different experiments were performed. First, antennal nerves 2–4 of the last abdominal ganglion that contain many of

J Neurophysiol • VOL 104 • AUGUST 2010 • www.jn.org ROSTRAL SENSORY EXCITATION OF THE LATERAL GIANT NEURONS 679

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1.5 mV VNC_rostral LG-R 2 ms LG-R FIG. 3. Single-pulse electrical stimula- VNC_caudal tion of the right protocerebral tract causes depolarizations in the ipsilateral LG. A:a VNC_caudal weak stimulus produces one measurable ac- tion potential in the VNC and produces only a small depolarization of LG (light gray traces). A 2nd interneuron is recruited with stronger electrical stimulation and can be seen propagating through the VNC, resulting A[ 1] in a larger depolarization of LG (dark gray traces). Higher stimulus intensity resulted in KAc activation of more descending units, produc- ing a larger PSP in LG (black traces). A1: the Downloaded from VNC_rostral same experiment using KAc-filled micro- electrodes instead of KCl-filled electrodes in a different animal. LG PSPs increase in number and size with increasing stimulus 1.5 mV voltage (light gray, dark gray, black traces) to the protocerebral tract. LG-R 2 ms jn.physiology.org

VNC_caudal on February 10, 2012 the primary afferents that excite the LGs (Antonsen et al. 2005; traces in Fig. 4B). The right antenna II nerve was stimulated Herberholz et al. 2002). One intracellular electrode was used to with hook electrodes (5 V, 0.5 ms), causing the resulting record LG membrane potentials on the ipsilateral side of the depolarization in the LG to overlap with the depolarization sixth abdominal ganglion and neural activity was monitored produced by current injection. The combined depolarization with a hook electrode placed on the ipsilateral BC. Electric was sufficient to produce an action potential in the LG, which stimulation (5.6 V, 0.3 ms) applied to the tail afferents was could be recorded with the second intracellular electrode and adjusted so that the resulting EPSP in the LG (7 mV) was close an extracellular recording electrode placed on the BC (black to firing threshold (dark gray traces in Fig. 4A). A stimulus traces in Fig. 4B). When suprathreshold current injection was applied to the right antenna II nerve (5 V, 0.3 ms) alone used to elicit an action potential in the LG and this current resulted in a small depolarization of the LG (light gray traces injection was combined with stimulation of the ipsilateral in Fig. 4A). When the onsets of the rostral and caudal stimuli antenna II nerve, the threshold for LG activation was always were timed so that their resulting inputs to LG overlapped, the reached earlier than with current injection alone (data not combined input from both sources was sufficient to bring the shown). LG above threshold (black traces in Fig. 4A; note that the LG action potential has been truncated). In these experiments, the Identifiable descending neurons produce EPSPs in LG antenna II nerve was stimulated several milliseconds before the sensory nerves of the tail to allow enough time for the descend- The BC was impaled near the brain with an intracellular ing units to reach the sixth abdominal ganglion and to coincide electrode to identify individual descending interneurons that with the peak of the EPSP received from caudal inputs. Control were activated by electrical stimulation of the antenna II nerve experiments with KAc-filled electrodes (n ϭ 2) produced the and produced EPSPs in the LG (Fig. 5). Several neurons that same results (Fig. 4A1). A stimulus to the right antenna II nerve matched both criteria were found in different preparations (n ϭ (5.0 V, 0.3 ms) caused a small depolarization in LG; when 14). Electrical stimulation of the antenna II nerve (3 V, 0.5 ms) combined with subthreshold stimulation of tail afferents (1.5 caused firing of many of the descending interneurons, some of V, 0.3 ms), the inputs summed and fired the LG neuron. which produced EPSPs in LG. In one such example, an action In a second set of experiments, subthreshold current (179 potential was recorded in one of these interneurons in the nA, 5 ms) was injected into the LG via a second KCl-filled ipsilateral brain connective 7 ms after the stimulation (Fig. 5A). intracellular electrode positioned in the same interganglion Prolonged current injection (50 nA, 20 ms) into the same segment and in close proximity to the recording electrode (gray interneuron caused it to fire multiple action potentials (Fig.

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FIG. 4. Descending inputs cannot alone cause action potentials in the LGs, but when paired with excitatory postsynaptic potentials (EPSPs) elicited by stimulation of tail afferents, the inputs sum to yield firing. A: a single electric stimulus applied to the right antenna II nerve resulted in a small depolarization of the LG (light gray traces; arrows). Electric stimulation applied to the tail afferents (arrowheads) was adjusted so that the resulting EPSP in the LG was close to firing threshold (dark gray traces). When rostral and caudal stimuli were timed so that their resulting inputs to LG overlapped, the combined input from both sources was sufficient to bring the LG above threshold (black traces; LG action potential truncated). A1: the results obtained with KCl-filled electrodes were replicated in different animals using electrodes filled with KAc. B: subthreshold current (179 nA, 5 ms) was injected into the LG via a 2nd intracellular electrode (gray traces). The right antenna II nerve was electrically stimulated causing the resulting depolarization in the LG to overlap with the depolarization produced by current injection. The combined depolarization was sufficient to produce an action potential in the LG that can be recorded with the 2nd intracellular electrode and an extracellular recording electrode placed on the BC (black traces).

5B). The spikes were observed in the extracellular electrodes several action potentials which produced corresponding EPSPs placed on the VNC, and they evoked corresponding unitary in the ipsilateral LG. When descending inputs from a single EPSPs in the ipsilateral LG (Fig. 5B). Injections of lower interneuron were paired with tail afferent inputs, the LG EPSP currents or for shorter time periods often caused single action increased in size (Fig. 5C). Activation of the BC interneuron potentials in individual interneurons and corresponding EPSPs with current injection (60 nA, 25 ms) produced a series of in LG. The temporal relationship between interneuron spikes small (0.5 mV) PSPs in LG (light gray trace). Stimulation of and LG EPSPs was very consistent for each individual inter- the A6 sensory nerve alone (0.7 V, 0.3 ms; dark gray trace) neuron but varied across preparations and according to LG also caused a PSP in LG (1.5 mV). When rostral and caudal recording site. The temporal delay measured between the peak inputs coincided, they summed to produce a larger EPSP in LG amplitudes of interneuron spikes elicited by current injection (2 mV; black trace). and corresponding LG EPSPs was 4.8 Ϯ 0.5 (SD) ms for recordings obtained in A4 (n ϭ 7) and 6.6 Ϯ 0.5 ms for DISCUSSION ϭ recordings obtained in A6 (n 5). Similar results were Escape behavior in crayfish obtained from experiments using microelectrodes filled with KAc (Fig. 5B1; n ϭ 2). Current injection (60 nA, 20 ms) into Fast and powerful escape tail-flips in crayfish are mediated a descending mechanosensory interneuron caused it to fire by neural circuits that consist of pairs of giant interneurons,

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FIG. 5. The BC was impaled near the brain with an intracellular electrode to identify individual descending interneurons that were activated by electrical stimulation of the antenna II nerve and produced EPSPs in the LG. A: electrical stimulation of the antenna II nerve caused firing of many of the descending interneurons, some of which produced EPSPs in LG, including one that can be seen in the intracellular recording trace of the ipsilateral brain connective shortly after the stimulation was applied to the antennal nerve. B: current injection into this interneuron caused it to fire multiple action potentials. The spikes can be seen in the extracellular electrodes placed on the VNC and evoke corresponding EPSPs in the ipsilateral LG.B1: the same results were obtained with KAc-filled electrodes instead of KCl-filled electrodes. C: descending inputs from a signal interneuron (light gray trace) were timed to coincide with ascending inputs from tail afferent stimulation (dark gray trace). The combined inputs produced a larger EPSP in LG (black trace). The experiment was performed using KAc-filled electrodes. giant motor neurons, and electrical coupling between these rear of the crayfish’s body. Using largely intact preparations of components. The lateral giant (LG) interneurons are the key juvenile crayfish, we have now shown for the first time that the components of the LG circuit, and it has previously been LG interneurons have more extensive receptive fields than assumed that they can only be excited by stimuli directed to the previously thought, and they receive excitatory inputs follow-

J Neurophysiol • VOL 104 • AUGUST 2010 • www.jn.org 682 Y.-C. LIU AND J. HERBERHOLZ ing rostral sensory stimulation. These descending inputs lower anosensory, and chemosensory, some of which are descending the threshold for LG-mediated escape tail-flips and are there- and some of which are ascending (Sandeman et al. 1992; fore likely to enhance the animal’s ability to respond adap- Sullivan and Beltz 2001). Although most of the axons found in tively. This is because any attack that is ultimately directed to the antenna II nerve belong to mechanosensory afferents (San- rear of the animal will be preceded by multimodal sensory deman 1989), and most of the large descending axons in the stimulation (e.g., visual and hydrodynamic) before the actual protocerebral tract (also referred to as the “optic tract” in the physical contact occurs, and if this information is detected by older literature) belong to visual interneurons (Cooper et al. widely distributed sensory systems and integrated with sensory 2001; Wiersma and Yamaguchi 1966, 1967), future experi- stimulation to the rear, it will enhance the animal’s chance of ments will have to use natural visual and natural tactile stimuli surviving. Moreover, during exploration of their environment for identification of specific sensory modalities that are respon- (e.g., during foraging), any “suspicious” signal detected by sible for the increase of the LGs’ excitability in the abdomen. antennal contact or by the eyes could lead to an increase of the LGs’ excitability and thus enhance the escape system’s sensi- Descending excitation versus depolarizing inhibition tivity for a possible attack. Such a mechanism has been shown in cockroaches where antennal palpation provides cues for Previously described descending inputs to the LGs that predator detection, and such textural information (rather than originate in the thoracic ganglia and brain were considered to chemical information) is used to modify the escape behavior be purely inhibitory; they provide “tonic” inhibition that mod- (Comer et al. 1994, 2003). ulates the LG’s threshold during specific behaviors such as feeding, restraint and possibly also during social interactions Downloaded from Organization of escape giant neurons’ receptive fields (Krasne and Edwards 2002b; Krasne and Lee 1988; Krasne and Wine 1975). Although descending inhibitory neurons have not After electrical stimulation of rostral sensory nerves, action been identified, tonic inhibition is thought to result from potentials were seen in the BCs within 10 ms after stimulation, GABA-mediated changes of chloride channel activity at inhib- and they arrived in the abdomen only a few milliseconds later. itory synapses that are located distally on the LG’s dendrites Thus information transfer is fast, and spikes that originate (Vu and Krasne 1992, 1993). Tonic depolarizing inhibition is rostrally arrive in the abdomen fast enough to affect caudal characterized by a persistent depolarization of LG without jn.physiology.org events that happen shortly after. The relatively short time it rapid fluctuations in membrane potential indicating very slow takes for action potentials to descend from rostral centers to the decay rates for IPSPs (Vu et al. 1993). Moreover, “recurrent” abdomen and to elicit EPSPs in the abdominal LGs, implies GABAergic inhibition of the LG neurons, which happens that the information is transmitted via individual descending proximal near the spike initiation zone, causes large depolar- interneurons that project from the brain to the last abdominal izing IPSPs that are much longer than the brief depolarizing segment and make monosynaptic connections with the LGs. events seen in our study (Roberts 1968; Vu and Krasne 1992). on February 10, 2012 The descending inputs from several activated interneurons can Thus long and persistent depolarizing IPSPs are in sharp summate and produce long-lasting compound EPSPs in the contrast to the short depolarizations seen in our experiments. LGs. Likewise, single interneurons that produce multiple The excitatory nature of these brief events is further evidenced spikes cause several milliseconds of depolarization in the LGs by the lower input needed from the tail afferents to fire the LGs thus providing an “open time window” during which subse- when caudal inputs coincide with rostral inputs. We performed quent excitatory inputs from tail afferents can combine with these experiments with both KCl- and KAc-filled electrodes this depolarization. Although we only recorded relatively small and obtained identical results; this strongly suggests that the LG EPSPs in response to rostral stimuli, we cannot exclude the LGs receive excitatory inputs from rostral sensory regions. possibility that summation of rostral inputs after repetitive How these excitatory inputs are integrated with inhibitory stimulation of multiple sensory channels are sufficient to fire inputs to orchestrate the most adaptive escape responses re- the LGs, especially in freely behaving animals. mains to be determined. Sensory stimulation of the protocerebral tract or the antenna II nerve also elicit EPSPs in the MGs; because the MGs receive Comparison to other escape systems their inputs in the brain, interneuronal activity in the BCs or nerve cord cannot be directly linked to MG EPSPs. However, Fast escape responses of decapods are typically controlled both MG EPSPs and LG EPSPs appeared with the first recruit- by neural circuits that contain LG and MG giant interneurons ment of descending units, and thus it is possible that the same (Espinoza et al. 2006). Given the many similarities in escape interneurons innervate the MGs and LGs. Another interesting behavior and underlying neural circuitry among these closely question is whether the MGs have receptive fields that extend related species, it is likely that our findings generalize to other to the abdomen and receive subthreshold excitatory inputs decapods with giant escape circuits. Escape responses have from this area. Preliminary data indicate that this may be the also been investigated in a number of noncrustacean model case (Herberholz and Liu 2008). More experiments toward this systems. Extensive work has been done in insects where escape end are planned for the future. behavior and underlying neural circuitry parallels the crayfish Using broad electrical stimulation of the antenna II nerve escape system to a certain extent. For example, cockroaches and the protocerebral tract, we were unable to unambiguously and crickets escape from wind stimuli by turning, running, determine the identity of activated sensory modalities. This is and/or jumping. Wind stimuli cause deflection of hairs located because the antenna II nerve contains both mechano- and on the cerci, and sensory afferents activate multiple giant chemosensory afferents, and the protocerebral tract contains interneurons that ascend from the abdomen to the thoracic axons of many sensory modalities, including visual, mech- region to initiate the escape maneuvers (Gras and Hörner 1992;

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2007, p. 71–89. jn.physiology.org Herberholz J, Antonsen BL, Edwards DH. A lateral excitatory network in GRANTS the escape circuit of crayfish. J Neurosci 22: 9078–9085, 2002. This work was in part supported by Research Grant IOS-0919845 from the Herberholz J, Edwards DH. The control of escape in crayfish through National Science Foundation. Y.-C. Liu received support from the National interactions of command neurons. Soc Neurosci Abstr 31: 754.7, 2005. Taiwan University and was additionally supported by a stipend from the Herberholz J, Issa FA, Edwards DH. Patterns of neural circuit activation and Psychology Department at the University of Maryland, College Park, MD. behavior during dominance hierarchy formation in freely behaving crayfish. J Neurosci 21: 2759–2767, 2001. Herberholz J, Liu Y-C. Receptive field organization of the giant escape on February 10, 2012 DISCLOSURES neurons in crayfish. Soc Neurosci Abstr 198.4, 2008. Herberholz J, Sen MM, Edwards DH. Escape behavior and escape circuit No conflicts of interest, financial or otherwise, are declared by the author(s). activation in juvenile crayfish during prey-predator interactions. J Exp Biol 207: 1855–1863, 2004. REFERENCES Kanou M, Ohshima M, Inoue J. The air-puff evoked escape behaviour of the cricket Gryllus bimaculatus and its compensational recovery after cercal Araki M, Nagayama T. Direct chemically mediated synaptic transmission ablations. Zool Sci 16: 71–79, 1999. from mechanosensory afferents contributes to habituation of crayfish lateral Krasne FB. Excitation and habituation of the crayfish escape reflex: the giant escape reaction. J Comp Physiol [A] 189: 731–739, 2003. depolarizing response in lateral giant fibers of the isolated abdomen. J Exp Araki M, Nagayama T. Decrease in excitability of LG following habituation Biol 50: 29–46, 1969. of the crayfish escape reaction. J Comp Physiol [A] 191: 481–489, 2005. Krasne FB, Edwards DH. Modulation of the crayfish escape reflex—physi- Antonsen BL, Edwards DH. Mechanisms of serotonergic facilitation of a ology and . Integr Comp Biol 42: 705–715, 2002a. command neuron. J Neurophysiol 98: 3494–504, 2007. Krasne FB, Edwards DH. Crayfish behavior: lessons learned. In: Crustacean Antonsen BL, Herberholz J, Edwards DH. The retrograde spread of synaptic Experimental Systems in Neurobiology, edited by Wiese K. New York: potentials and recruitment of presynaptic inputs. J Neurosci 25: 3086–3094, Springer, 2002b, p.3–22. 2005. Krasne FB, Lee SC. Response-dedicated trigger neurons as control points for Blake MA, Hart PJB. The vulnerability of juvenile signal crayfish to perch behavioral actions: selective inhibition of lateral giant command neurons and eel predation. Freshwater Biol 33: 233–244, 1995. during feeding in crayfish. J Neurosci 8: 3703–12, 1988. Burdohan JA, Comer CM. An antennal-derived mechanosensory pathway in Krasne FB, Teshiba TM. Habituation of an invertebrate escape reflex due to the cockroach: descending interneurons as a substrate for evasive behavior. modulation by higher centers rather than local events. Proc Natl Acad Sci Brain Res 535: 347–352, 1990. USA 92: 3362–3366, 1995. Comer CM, Parks L, Halvorsen MB, Breese-Terteling A. The antennal Krasne FB, Wine JJ. Extrinsic modulation of crayfish escape behavior. J Exp system and cockroach evasive behavior. II. Stimulus identification and Biol 63: 433–50, 1975. localization are separable antennal functions. J Comp Physiol [A] 189: Liden WH, Herberholz J. Behavioral and neural responses of juvenile 97–103, 2003. crayfish to moving shadows. J Exp Biol 211: 1355–1361, 2008. Comer CM, Mara E, Murphy KA, Getman M, Mungy MC. Multisensory Lima SL, Dill LM. Behavioral decisions made under the risk of predation: a control of escape in the cockroach Periplaneta americana. II. Patterns of review and prospectus. Can J Zool 68: 619–640, 1990. touch-evoked behavior. J Comp Physiol 174: 13–26, 1994. Mellon D. Convergence of multimodal sensory input onto higher-level neu- Cooper RL, Li H, Long LY, Cole JE, Hopper HL. Anatomical comparisons rons of the crayfish olfactory pathway. J Neurophysiol 84: 3043–3055, 2000. of neural systems in sighted epigean and troglobitic crayfish species. J Mittenthal JE, Wine JJ. Connectivity patterns of crayfish giant interneurons: Crustacean Biol 21: 360–374, 2001. visualization of synaptic regions with cobalt dye. Science 179: 182–184, Correia AM. Seasonal and interspecific evaluation of predation by mammals 1973. and birds on the introduced red swamp crayfish Procambarus clarkii Newland PL, Aonuma H, Nagayama T. Monosynaptic excitation of lateral (Crustacea, Cambaridae) in a freshwater marsh (Portugal). J Zool 255: giant fibers by proprioceptive afferents in the crayfish. J Comp Physiol [A] 533–541, 2001. 181: 103–109, 1997.

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