Cholinergic Neurotransmission from Mechanosensory ... - BioOne

© 2008 Zoological Society of Japan

ZOOLOGICAL SCIENCE 25: 517–525 (2008)

Cholinergic Neurotransmission from Mechanosensory Afferents to Giant Interneurons in the Terminal Abdominal Ganglion of the Cricket Gryllus bimaculatus Oak Yono and Hitoshi Aonuma* Laboratory of Neuro-Cybernetics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan

Crickets respond to air currents with quick avoidance behavior. The terminal abdominal ganglion (TAG) has a neuronal circuit for a wind-detection system to elicit this behavior. We investigated neuronal transmission from cercal sensory afferent neurons to ascending giant interneurons (GIs). Pharmacological treatment with 500 μM acetylcholine (ACh) increased neuronal activities of ascending interneurons with cell bodies located in the TAG. The effects of ACh antagonists on the activities of identified GIs were examined. The muscarinic ACh antagonist atropine at 3-mM concentration had no obvious effect on the activities of GIs 10-3, 10-2, or 9-3. On the other hand, a 3mM concentration of the nicotinic ACh antagonist mecamylamine decreased spike firing of these interneurons. Immunohistochemistry using a polyclonal anti-conjugated acetylcholine antibody revealed the distribution of cholinergic neurons in the TAG. The cercal sensory afferent neurons running through the cercal nerve root showed cholinergic immunoreactivity, and the cholinergic immunoreactive region in the neuropil overlapped with the terminal arborizations of the cercal sensory afferent neurons. Cell bodies in the median region of the TAG also showed cholinergic immunoreactivity. This indicates that not only sensory afferent neurons but also other neurons that have cell bodies in the TAG could use ACh as a neurotransmitter. Key words:

acetylcholine, nicotinic ACh receptor, immunohistochemistry, insects, cercal system

INTRODUCTION A wind-detective mechanosensory processing system is thought to be the principal mechanism controlling avoidance behavior in crickets. Neuronal mechanisms underlying this behavior have been well investigated (Mendenhall and Murphey, 1974). Air currents are detected by mechanoreceptive hairs (filiform hairs) arranged on the surface of appendages called cerci at the rear of the cricket’s abdomen. Movement of these filiform hairs initiates spikes of sensory afferent neurons. The sensory afferent neurons project into the cricket terminal abdominal ganglion (TAG). Many neurons in the TAG have been identified by their morphological and physiological characteristics (Jacobs and Murphey, 1987; Baba et al., 1991). Especially, the giant interneurons (GIs) receive excitatory inputs directly from the sensory afferent neurons to activate the avoidance behavior program (Edwards and Palka, 1974; Palka et al., 1977; Gnatzy and Tautz, 1980). These neuronal circuits must have a minimum time frame to avoid threats such as predatory digger wasps (Gnatzy and Hustert, 1989; Gnatzy and Kämper, 1990; * Corresponding author. Phone: +81-11-706-3832; Fax : +81-11-706-4971; E-mail : [email protected] doi:10.2108/zsj.25.517

Hirota et al., 1993; Baba and Shimozawa, 1997). Meyer and Reddy (1985) demonstrated that putative nicotinic acetylcholine (ACh) receptors are localized in synaptic regions of the well-characterized GI pathway in the TAG. However, immunohistological evidence of the localization of ACh in the sensory afferent neurons remains lacking. ACh is one of the neurotransmitters in the central nervous system. In general, ACh is a major neurotransmitter released from sensory afferent neurons in invertebrates. Nicotinic (e.g., Sattelle, 1980; Benke and Breer, 1989) and muscarinic (e.g., Leitch and Pitman, 1995) receptors of ACh have been found in insects. Nicotinic receptors on the postsynaptic neuron mediate fast excitatory neurotransmission from sensory afferent neurons in the insect central nervous system (Sattelle, 1980). Insects have reflex circuits in their nervous system to monitor their environment continuously and to avoid potential threats. Fast excitatory neurotransmission must be one of the most important neuronal mechanisms in reflex behavior, particularly in escape and avoidance behavior that is evoked by a specific set of stimuli. A nicotinic ACh receptor required for escape behavior has been found in Drosophila (Fayyazuddin et al., 2006). Cholinergic neurons have been also demonstrated by use of immunohistochemistry with an antiserum against choline acetyltransferase in the fruit fly, Drosophila melanogaster (Yasuyama et al., 2002). To identify neurotransmitters, it is necessary to demonstrate that a candidate substance is present in the presyn-

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aptic neuron to be released, and that the receptor of the transmitter candidate presents at postsynaptic neurons. However, principal evidence of the localization of ACh in the sensory afferent neurons is lacking for the GI pathways in the cricket TAG. The aim of this study was to demonstrate cholinergic neurons in the cricket TAG using immunohistochemical techniques. In particular, we focused on sensory afferent neurons terminating in the TAG. Our results suggest that not only sensory afferent neurons but also other neurons with cell bodies in the TAG use ACh as a neurotransmitter. MATERIALS AND METHODS Animals All experiments were performed using adult male crickets, Gryllus bimaculatus (DeGeer), that were reared in plastic cases (80 cm×45 cm×20 cm) on a 14 h:10 h light-dark cycle at 27±2°C. Animals were fed a diet of insect food pellet, chopped carrots, and water. Male crickets in this study were used within 2 weeks after molting. Electrophysiological and pharmacological experiments Crickets were pinned dorsal side up on a cork holder after the head, legs, and wings were removed. The haemolymph of the crickets usually coagulated, and the cut end of the thorax was filled. A thin flap of dorsal cuticle, gut, and fat bodies were gently removed to expose the abdominal connectives, and the body cavity was filled with normal cricket saline (140 mM NaCl, 10 mM KCl, 1.6 mM CaCl2, 2 mM MgCl2, 44 mM Glucose, 2 mM TES, pH 7.2). Wind stimulation was applied to the preparation in the center of a wind tunnel (inner diameter 45 mm, length 140 mm). During the experiment, filiform hairs on the surface of cerci were stimulated by air currents generated by loudspeakers. The detailed methods of stimulation are described in Kanou and Shimozawa (1984). Briefly, loudspeakers were attached at both ends of the wind tunnel, and a Gaussian white noise (GWN) waveform was generated by low-pass filtering of a pseudo-random binary msequence (CG-742N, NF Corporation) through an attenuator (STA113, Tokyo Kouon Denpa) with push-and-pull mode (Fig. 1). The velocity profile of the wind stimulation was set at a bandwidth of 500 Hz and root mean square (RMS) amplitude of 10 mm/s, since this stimulation initiates spikes in the GIs. By using tungsten hook electrodes, we made extracellular recordings of the GIs from the connectives between the 6th abdominal ganglion and TAG. All physiological data with stimulus waveforms were stored in a digital data recorder (PC208Ax, Sony) at a sampling rate of 48 kHz for later analysis. To examine the cholin-

Fig. 1. Diagram of the experimental setup. Air currents were generated by loudspeakers attached on both ends of the wind tunnel. The wind tunnel could be rotated to change the direction of the air-current stimulation. Neuronal responses were recorded using an intracellular electrode and/or extracellular electrodes during the stimulation.

ergic connection between sensory afferent neurons and GIs pharmacologically, we here focused on three different types of GIs: 103, 10-2, and 9-3 (nomenclature according to Jacobs and Murphey, 1987). The activities of thse GIs were recorded in the neuropil of the TAG by using a microelectrode intracellularly and simultaneously from connectives by using hook electrodes extracellularly. The extracellular spikes were sorted into each neuron on the basis of their amplitudes and shapes (Lewicki, 1998). Each sorted neuron was classified by physiological criteria such as the directional tuning curve and the first-order Wiener kernel (Kondou et al., 1993). The criteria were confirmed by simulataneous intracellular and extracellular recordings. The tuning curves of these GIs were in good agreement with those reported previously (Miller et al., 1991). Pharmacological treatments were performed to examine cholinergic neurotransmission between sensory afferent neurons and GIs. Atropine and mecamylamine were used at 3-mM concentrations as muscarinic and nicotinic ACh antagonists, respectively. Each agent was dissolved in normal cricket saline. The saline containing each agent was bath applied for 6 min and then quickly washed out with fresh normal saline. In some preparations, 500 μM ACh was bath applied and the responses of ascending interneurons were examined. All pharmacological agents were obtained from Sigma Chemical Co. Histological experiments Immunohistochemical staining of ACh in the form of proteinconjugated choline was carried out using a protocol modified from Leitinger and Simmons (2000). Animals were anaesthetized by rapid cooling on crushed ice. Cricket TAGs were then dissected out in cooled cricket saline, and fat bodies and trachea were gently removed. Preparations were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 1% of sodium metabisulfite (SMB) at 4°C overnight. After fixation, they were treated with 0.5% of sodium borohydride (NaBH4, Sigma) in 0.05 M Tris-HCl buffer containing 0.45% SMB (Tris-HCl SMB), pH 7.4, for 20 min. After rinsing with Tris-HCl SMB buffer (4×20min), the preparations were washed with Tris-HCl SMB buffer containing 0.5% Triton X-100 (TX, Sigma) for 1–2 hr. The preparations were then incubated with 5% normal goat serum diluted in Tris-HCl SMB buffer containing 0.5% Triton X-100 for 2–3 hr. Afterward, they were incubated with a polyclonal anticonjugated acetylcholine antibody solution (1:1000, diluted in TrisHCl SMB; MoBiTec) for 48 hrs at 4°C. Preparations were then washed for 2 hr in 0.05 M Tris-HCl buffer containing 0.5% Triton X100. Control preparations were incubated without the primary or secondary antibody. These preparations did not show any immunoreactivity. As a further control, preadsorption tests were performed. After the primary antibody (1:1000, 100 μl) was preincubated with antigen containing 2 mg ACh for 4 hr at room temperature, it was centrifuged at 10,000×g for 15 min, and the supernatant was collected. Control preparations were incubated in the supernatant for 48 hr at 4°C and then washed for 2 hr in 0.05 M Tris-HCl buffer containing 0.5% Triton X-100. To visualize the cholinergic neurons, goat anti-rabbit Cy3-conjugated IgG (1:500 in 0.05 M Tris-HCl buffer containing 0.5% TX) was applied as the secondary antibody at 4°C overnight. The preparations were then washed in phosphate-buffered saline (PBS), dehydrated with an ethanol series, and cleared in methyl salicylate. The terminal arborizations of sensory afferent neurons innervating filiform hairs were examined by using neurobiotin staining. After removal of the head and legs, crickets were pinned on a Sylgard-lined Petri dish. The cerci of the cricket were cut at the root, and the cut ends of the cerci were filled with a 5% solution of neurobiotin (VECTOR) in distilled water. Preparations were incubated at 4°C overnight, and the TAG was removed from the abdomen and fixed with 4% paraformaldehyde for 3 hr. Preparations were then washed with PBS containing 0.5% Triton X-100 (PBSTX) for 2 hr with agitation. They were visualised by using Cy3 conjugated streptavidin (ZYMED) diluted with PBSTX (1:200). After incubation in the secondary antibody solu-

Cholinergic Neurons in the Cricket TAG tion at 4°C for 2 days, they were washed with PBS for 2 hr, dehydrated by using an ethanol series, and cleared with methyl salicylate. Stained preparations were observed under a confocal scanning microscope (FV-300, Olympus), and all images recorded were stored as TIF-format files for later analysis.

RESULTS Response of ascending interneurons to air-current stimulation Constant air-puff stimulation initiated spikes in ascending interneurons (Fig. 2A). Intracellular and extracellular recordings were simultaneously performed to examine the physiological characteristics of an ascending interneuron (Fig. 2B). Extracellular spikes were followed by intracellular spikes within short latency. These spikes were judged to be recorded from the same neuron. For example, the response of the identified GI 10-3 to the air-current stimulation is shown in Fig. 2B. Some of the GIs could be identified by their physiological characteristics. In this study, we focused on GIs 10-3, 10-2, and 9-3. These GIs initiated large, different-sized spikes when filiform hairs were stimulated by air currents. The sources of these spikes could be identified from physiological properties, such as for GI 10-3, which responded to air currents from the anterior direction. Directional tuning curves were made from the extracellular recordings (Fig. 3). The right side of GI 10-3 (10-3R, n=11) responded to air currents

Fig. 2. Response of ascending interneurons to air-current stimulation. (A) Extracellular recording of the response of ascending interneurons to constant air-puff stimulation. The stimulation initiated spikes in ascending interneurons. Extracellular spikes were recorded from the connective nerve between the 6th abdominal ganglion and the TAG. (B) Response of identified GI 10-3 to GWN air-current stimulation. The GWN air current initiated spikes in GI 10-3 (indicated by asterisks). Intracellular and extracellular recordings were performed simultaneously. Intra rec, intracellular recording; extra rec, extracellular recording; stim, monitor of air-current stimulation.

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within an approximately 45° range at the right anterior part of the cricket. The left side of GI 10-3L (n=11), on the other hand, responded to air currents within a 45° range at the left anterior part of the cricket (Fig. 3A). The tuning curves of GIs 10-2 and 9-3 were also examined. GIs 10-2R and 102L responded to the air current within 45° ranges at the right and left posterior parts of the cricket, respectively (Fig. 3B). GI 9-3 responded to air currents from a wide range in the lateral parts of the crickets (Fig. 3C). These results provided us criteria to identify, from extracellular recordings, which GIs initiate spikes by a particular air stimulation. Pharmacological experiments Cercal sensory afferent neurons must release neurotransmitters upon air-current stimulation. Cholinergic neurotransmission from sensory afferent neurons to ascending interneurons was examined by pharmacological experiments (n=9). Bath application of ACh increased spike activities of interneurons recorded from the connective nerves between the TAG and the 6th abdominal ganglion (Fig. 4). The effect of ACh on the spike activities of ascending interneurons appeared to be phasic. When ACh was applied, the frequency of the major spikes of some GIs increased. After 2

Fig. 3. Air-direction-specific responses of identified GIs. Directional tuning curves in terms of spike firing rate are plotted on polar coordinates (mean±SD). The body axis is aligned on 0-180 degrees. Zero degrees represents the cricket head; 90°, the right side of the cricket; 180°, the posterior; and 270°, the left side of the cricket. (A) Directional tuning curves of the GI 10-3. The left side of GI (10-3L) responded to the GWN air-current stimulation from the anterior side, in particular, within a 45° range on the left side. The opposite side of the GI (10-3R) responded mainly to air currents from a 45° range on the right side. (B) Directional tuning curves of GI 10-2. GIs 10-2R and 10-2L tuned the right posterior and left posterior parts of the cricket, respectively. (C) Directional tuning curves of GI 9-3. GIs 9-3R and 93L tuned the right and the left sides of the cricket, respectively.

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Fig. 4. Effect of bath application of ACh on the spike activities of ascending interneurons originating in the TAG of a cricket. The activities of interneurons were recorded at the connective nerve between the 6th abdominal ganglion and the TAG. The frequency of the major spikes was counted. Cricket saline containing 500 μM ACh was bath applied for 2 min. The frequency of the spikes increased just after the application of Ach, then gradually decreased to the control level.

min of application, the spike frequencies decreased slightly. After ACh solution was replaced by fresh normal saline, the activities of major spikes recovered to the original level. Similar results were observed for independent preparations (n=8). This suggested that the response of ascending interneurons to ACh mimics that for air-current stimulation. The receptor types of identified GIs were next examined by applications of the atropine inhibitor of the muscarinic ACh receptor, and the mecamylamine inhibitor of the nicotinic ACh receptor (Fig. 5). GIs 10-3, 10-2 and 9-3, which receive monosynaptic inputs from cercal sensory afferent neurons (Chiba et al., 1992; Killian et al., 1993), were identified by using the criteria shown in Fig. 3. The effects of antagonists were examined by observing extracellular spikes. Extracellular recordings were performed during the GWN air-current stimulation as antagonists of ACh receptors were applied. Atropine was bath applied during the aircurrent stimulation, and the spike activities of identified GIs 10-3 (n=7), 10-2 (n=8) and 9-3 (n=6) were recorded (Fig. 5A). Even after application of atropine, spike frequencies did not change obviously. On the other hand, bath application of mecamylamine, a nicotinic ACh receptor antagonist, gradually decreased spike firing in GIs 10-3 (n=10), 10-2 (n=11), and 9-3 (n=6), resulting in spikes that were almost absent (Fig. 5B). After washing with fresh normal saline, spike activities recovered to about 80% of original activities. Cholinergic immunoreaction in the TAG The pharmacological experiments suggested cholinergic neurotransmission in the cercal system in the TAG (Figs. 4, 5). Immunohistochemical staining for ACh was then performed to confirm the distribution of cholinergic neurons in the TAG. The neuropil region, where mechanosensory afferents from the cercal nerve terminate, was densely stained by the cholinergic immunohistochemistry (Fig. 6). Cholinergic immunoreactivity was observed in the cercal sensory afferent neurons at the cercal nerve root (indicated by arrow-

Fig. 5. Effect of antagonists of ACh receptors on the spike frequencies of GIs. The spike frequency was normalized to the spike frequency before the bath application. (A) Effect of bath application of 3 mM atropine on the spike frequency of identified GIs 10-3, 10-2, and 9-3. Neuronal activities were recorded extracellularly from the left and right sides of connective nerves between the TAG and the 6th abdominal ganglion. Atropine was applied for 6 min (indicated by thick bar). The frequency of spike firing (mean±SE) in each neuron did not change with the application of the solution containing atropine. (B) Effect of mecamylamine on the spike frequency of the identified interneurons. Bath application of 3 mM mecamylamine significantly decreased the spike frequency of the neurons to almost zero (mean±SE; GI 10-3, p=0.0001; GI 10-2, p=0.0004; GI 9-3, p=0.01; t-tests between spike activity at 180 sec and that at 720 sec); with fresh normal saline, spike frequency partly recovered.

heads in Fig. 6A, B). The neuropil region around the middle and dorsal parts of the TAG also showed strongly cholinergic immunoreactivity (Fig. 6C, D). The median and posterior regions of the ventral horizontal plane showed strong cholinergic immunoreactivity, whereas the posterior ventral commissure failed to show immunoreactivity. The dorsal plane of the TAG neuropil also showed strong immunoreactivity. As a control, we performed three different sets of experiments. When the primary or secondary antibody was omitted from the staining processes, no neurons showed any immunoreactive signals (data not shown). Control preparations used in the preabsorption test did not show any obvious immunoreactive signals (Fig. 6E, F). These control experiments support the specificity of the antibody against ACh (Figs. 4, 5). Mechanosensory afferent neurons run through the cercal nerve into the neuropil of the TAG. The terminal arborizations of the cercal sensory afferent neurons were observed (Fig. 7). Some of the cercal sensory afferent neurons expanded their branches in the median-distal region of

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Fig. 6. Cholinergic immunoreactivity in the cricket TAG. The neuropil region where sensory afferent neurons projected neuronal branches showed strongly cholinergic immunoreaction. (A) Confocal images at the horizontal plane around 100 μm from the ventral surface of the TAG. Five stacks of images taken at 3-μm intervals were merged. The arrowhead shows the tract of sensory afferent neurons innervated from the cercal nerve root. (B) High magnification of the stained region indicated by the arrowhead in image A. The neuronal tract came from the neuropil and ran into the cercal nerve root (shown by arrowhead). (C) Stained neuropil at around 200-μm depth from the ventral surface of the TAG. Five stacks of images taken at 3-μm intervals were merged. The arrowhead indicates the posterior ventral commissure (PVC). (D) Stained neuropil at around 300-μm depth from the ventral surface of the TAG. Five stacks of images taken at 3-μm intervals were merged. (E, F) A control experiment showed no obvious immunoreactive signals in the TAG. When the primary antibody was preabsorbed by Ach, no neuronal cell bodies were stained at the ventral surface of the TAG (E). In addition, no immunoreactive signals were observed in the horizontal plane at around 100 μm from the ventral surface of the TAG (F). No cell bodies were stained in the lateral region of the ganglion, although A–D show some densely stained cell bodies. Scale bar indicates 100 μm.

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Fig. 7. Terminal arborizations of cercal sensory afferent neurons in the TAG. One-half of the TAG is shown. Sensory afferent neurons were stained using neurobiotin that was visualized by Cy3 conjugated streptavidin. (A) Confocal image of sensory afferent neurons in the horizontal plane at around 100 μm from the ventral surface of the TAG. Five stacks of images taken at 3-μm intervals were merged. (B) Stained sensory afferent neurons at around 200-μm depth from the ventral surface of the TAG. Five stacks of images taken at 3-μm intervals were merged. (C) Stained sensory afferent neurons at around 300-μm depth from the ventral surface of the TAG. Ten stacks of images taken at 3-μm intervals were merged. Scale bar indicates 100 μm.

the neuropil (Fig. 7A), some of them expanded in the medial part of the neuropil (Fig. 7B), and others expanded approximately in the rostral region of the neuropil (Fig. 7C). These terminal arborizations of the cercal sensory afferent neurons were similar to the immunoreactive staining pattern in the neuropil shown in Fig. 6. The focal planes of Figs. 7A, 7B, and 7C roughly correspond with those of Fig. 6A, Fig. 6C, and Fig. 6D, respectively. Many neuronal cell bodies in the TAG also showed positive immunoreaction (Fig. 8). Cell bodies about 40 μm in diameter that showed a positive immunoreaction were located mainly around the median ventral region of the TAG (Fig. 8A). On the dorsal side of the TAG, several cell bodies 40 μm in diameter were also found (Fig. 8B). The ventral median cells with a positive signal were divided into three groups (Fig. 8C, D). The ventro-anterior cell group had 2–4 cell bodies, the ventro-medial cell group had 5 or 6 cell bodies, and the ventro-distal cell group had 13 or 14 cell bodies. Smaller cell bodies (20 μm in diameter) were found around the ventro-medial region and the ventro-lateral region of the ganglion (Fig. 8E). In the dorso-distal region, several cell bodies showed positive immunoreaction. In the dorso-lateral region, about 10 cell bodies had a positive signal (Fig. 8F). DISCUSSION Cholinergic neurotransmission in the cercal system The spikes of wind-receptive sensory afferent neurons

activate the cricket GIs. GIs 10-3, 10-2, and 9-3 could be identified using the physiological criteria outlined in this study. GI 10-3 responds to air stimulation from the front side of the animal, whereas GI 10-2 responds to air stimulation from behind and GI 9-3 responds to air stimulation from the lateral side. It is expected that homologous neurons would share a similar function among different cricket species. Indeed, the results described here using G. bimaculatus are consistent with a previous study using A. domesticus (Miller et al., 1991), although in that study directional tuning curves were determined using a half-period sinusoidal stimulus of 2 Hz. To identify the neurotransmitter released from cercal sensory afferent neurons, we first applied ACh to the TAG. The effect of ACh on the spike activities of ascending interneurons mimicked air-current stimulation. Bath application of ACh on the TAG increases the Ca2+ concentration in the TAG (Ogawa, personal communication). Moreover, the identified GIs innervated by cercal sensory afferent neurons are known to exhibit an increase in Ca2+ concentration when they perceive air currents (Ogawa et. al., 1999, 2001, 2004). ACh could therefore be released from cercal sensory afferent neurons. GIs are an important component of the neuronal circuit to evoke fast avoidance behavior. Fast neurotransmission to introduce reflex behavior is usually mediated by nicotinic ACh receptors on the postsynaptic neuron (Sattelle, 1980). ACh receptor types of the GIs were then examined by application of atropine and mecamylamine. Atropine had no

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Fig. 8. Cholinergic immunoreactive cell bodies in the TAG. (A) Stained cell bodies were located on the ventral side of the TAG. Stained cell bodies about 40 μm in diameter were located in the median region of the TAG; these belong to the ventro-anterior (VA), ventro-median (VM), and ventro-distal (VD) cell groups. Stained cell bodies about 20 μm in diameter were located in the median (VM and VD) and lateral (VL, ventro-lateral cell group) regions of the TAG. (B) Several cell bodies about 40 μm in diameter were stained in the dorso-distal region. The lateral cell group was also stained (DL, dorso-lateral cell group). (C) High magnification of the area between VA and VM in image A. (D) High magnification at VD in image A. (E) High magnification at VL in image A. These stained cell bodies were located at around 100-μm depth from the ventral surface of the ganglion. (F) High magnification in the lateral region at DL in image B. These stained cell bodies were located at around 200-μm depth from the ventral surface of the ganglion. Scale bar in each image indicates 100 μm.

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obvious effects on spike activities initiated in GIs 10-3, 102, or 9-3, but mecamylamine abolished spike firing in GIs 10-3, 10-2, and 9-3 (Fig. 5). It is known that GIs in the TAG receive monosynaptic inputs from cercal sensory afferent neurons (A. domesticus: Chiba et al., 1992; Killian et al., 1993). The major fast excitatory neurotransmission from cercal sensory afferent neurons is thus likely mediated by ACh acting through nicotinic receptors on the GIs. The results of the pharmacological analyses in this study were similar to previous work done in A. domesticus by Meyer and Reddy (1985). The terminal arborizations of the cricket cercal sensory afferent neurons have been previously investigated (Bacon and Murphey, 1984). These terminal arborizations indicate an excitatory receptive field in the TAG, and the wind direction must be encoded by neurons in the TAG. An important mechanism for sensing wind direction is thus expected to be the synchronous activity of neurons. Indeed, specific pairs of GIs fire synchronously in response to a specific wind direction (Yono and Shimozawa, 2005). Short-term neuron-plasticity such as depression must also be important for sensing the wind direction. Short-term synaptic facilitation and depression have been found in identified GIs in a cricket (Killian and Murphey, 1998). Short-term depression might help to tune the sensitivity of the neuronal circuit for air detection. Synaptic depression is usually meditated by a chemical synapse. Therefore, cholinergic neurotransmission could mediate synaptic plasticity in the cercal system of the TAG. ACh immunoreactive neurons in the TAG The distribution of cholinergic-immunoreactive neurons in the cricket TAG was revealed by immunohistochemical staining with a polyclonal anti-conjugated acetylcholine antibody, the same approach that Geffard et al. (1985) used to visualize ACh in the locust brain. We were unable to clearly distinguish Ach-containing neurons from phosphatidyl choline-containing neurons in our study; neurons rich in phosphatidyl choline might have been stained. Phosphatidyl choline is used to produce ACh in cholinergic neurons. To examine whether cholinergic immunoreactive neurons release ACh or not, we performed pharmacological experiments using agonists of ACh receptors in cercal systems in the TAG. We demonstrated nicotinic neuron transmission from mechanosensory afferents to GIs and carried out preabsorption tests demonstrating that no neurons obviously showed immunoreaction. These results support specific immunoreaction in the cricket central nervous system. Cholinergic immunoreactivity was strongly observed in the neuropil of the cricket TAG. The distribution of the stained region of the neuropil overlapped with the distribution of the terminal arborizations of the cercal sensory afferent neurons. Histochemical evidence of cholinergic neurotransmission of the sensory afferent neurons was demonstrated using immunohistochemistry against the enzyme acetylcholine esterase. Immunoreactivity was found at the synaptic clefts around the afferent synapses (e.g., Rind and Leitinger, 2000). However, no direct evidence for ACh localization in the mechanosensory afferent neurons was found. In this study, we showed ACh-like immunoreactive neurons in the TAG, which provides further understanding of cholinergic neurotransmission in the TAG of the cricket.

ACh is known to mediate both excitatory and inhibitory responses. In insects, at least two types of ACh receptors have been found, nicotinic and muscarinic. Nicotinic type receptors mediate fast neurotransmission. A subclass of muscarinic ACh receptors is thought to mediate the slower inhibitory actions of ACh (muscarinic type 2; for review see Trimmer, 1995). Some neurons have mixed ACh receptors, for example, DUM neurons in the TAG of the cockroach (Grolleau et al., 1996). Our immunohistochemical results will help understand cholinergic neurotransmission in the cricket TAG. Indeed, little is known about cholinergic neurons except for sensory afferent neurons in the cricket TAG (e.g., Meyer and Reddy, 1985). We found here that not only sensory afferent neurons but also other kinds of neurons that have cell bodies in the TAG show cholinergic immunoreaction. This suggests that cholinergic neurotransmission mediates some kind of neuronal processing besides mechanosensory processing. The cricket TAG is well known as a center for controlling a variety of behaviors such as avoidance behavior using wind-detection systems (Mendenhall and Murphey, 1974), copulation behavior (Snell and Killian, 2000), and egg laying behavior (Sugawara and Loher, 1986). Many neuronal cell bodies in the TAG showed positive cholinergic immunoreactivity (Fig. 8). The TAG is thought to be composed of five ganglia, the 7th to 11th abdominal ganglia (Doe and Goodman, 1985). Some of the ventral-side cell bodies stained seemed to be located at each developmentally original ganglion. This suggests that these neurons could participate in segmental movement, like the circulatory system of insects. Smaller sizes of cell bodies were found around the medial and lateral regions of the ganglion. On the dorsal surface of the TAG, cell bodies were also stained. In invertebrates, ACh is known to be a neurotransmitter of interneurons in the central nervous systems. In crayfish, some motor neurons in the thoracic and abdominal nervous systems are activated by ACh (Yoshino et al., 1984; Cattaert et al., 1994). Casagrand and Ritzmann (1992) demonstrated that a population of thoracic interneurons receives direct inputs from a population of ventral GIs in the cockroach and that these synaptic transmissions are cholinergic. At present, we have not identified the cholinergic immunoreactive neurons that have cell bodies in the TAG. To identify the stained neurons, it is necessary to perform double labeling with intracellular staining and immunohistochemistry (Aonuma and Nagayama, 1999; Seki et al., 2005). Further investigation is necessary to fully understand cholinergic neurotransmission in the neuronal circuits controlling a variety of behaviors. ACKNOWLEDGMENTS We thank Dr. Derek B. Goto (Hokkaido University) for critical reading of this manuscript. We also thank Drs. H. Ogawa and T. Shimozawa for giving us constructive comments on this work. This work was supported in part by Grants-in-Aid from JSPS to H. A. (14704004) and from the Japanese Ministry of Education, Culture, Sports, Science and Technology for Scientific Research on Priority Areas (No. 454) to H. A. (17075001).

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