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1Department of Biology, Tokyo Gakugei University; and 2Tokyo Gakugei University Senior High School, Tokyo, Japan
Submitted 11 April 2008; accepted in final form 5 June 2008
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ABSTRACT |
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INTRODUCTION |
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; Schultz et al. 1993; Wise 2004). On the other hand, OA is found only in trace amounts and its function as a true neurotransmitter is unclear in vertebrate. In insect, however, there have been increasing evidences showing that OA mediates unconditioning stimulus (US) or reward (appetitive) signals, whereas DA mediates punishment (aversive) signals in olfactory learning. OA has been widely distributed in the insect nervous system and its roles are often compared with adrenergic receptors of vertebrates (Evans 1985). In honeybee, OA acts as a mediator that conveys sugar information, whereas DA acts as a mediator that conveys electrical shock information. Behavioral and pharmacological experiments with honeybee further revealed that OA injected into the mushroom bodies facilitates olfactory learning and memory retrieval (Bicker and Menzel 1989; Menzel 1990). OA has been shown to be released by ventral unpaired median (VUM) neuron onto antennal lobe and also mushroom body neurons and electrical stimulation of VUM neuron could substitute the US in the proboscis reflex (Kreissl et al. 1994). Hammer and Menzel (1998) provided further evidence that local injection of OA into the mushroom body calyces produces a lasting pairing-specific enhancement of proboscis reflex. Recently, Matsumoto and Mizunami (2000, 2002, 2004) and Matsumoto et al. (2006) have shown that crickets (Grylus bimaculatus) have high capacities to form olfactory long-term memory. By using both the behavioral and pharmacological techniques, Unoki et al. (2005, 2006) have demonstrated that OA and DA could mediate the reward and punishment signals, respectively, in both olfactory and visual learning. It has also been suggested that the mushroom body intrinsic neurons, called Kenyon cells, are the site of association of conditioning stimulus (CS) and US for both aversive and appetitive conditioning (Schwaerzel et al. 2003) and acetylcholine (ACh) has been postulated to convey CS (olfactory information) from the antennal lobes to the mushroom body Kenyon cell (Kreissl and Bicker 1989). Despite the presence of many studies describing that mushroom body Kenyon cells are the site of CS–US association, few studies have been carried out to investigate the action of putative neurotransmitters mediating CS and US signals and their signaling mechanisms in Kenyon cells. To understand the molecular basis of CS–US association underlying the olfactory learning, it is first necessary to characterize the target molecules—including receptors and ion channels expressed in native Kenyon cells—and then to investigate their modulation by putative neurotransmitters that convey US and CS information. The voltage-dependent ionic channels of Kenyon cells have been previously described (Cayre et al. 1998; Goldberg et al. 1999; Grünewald 2003; Grünewald et al. 2004; Schäfer et al. 1994; Wright and Zhong 1995). Ionotropic receptors such as ACh and 
-aminobutyric acid receptors have also been identified in cultured Kenyon cells (Grünewald et al. 2004; Su and Dowd 2003; Wright and Zhong 1995). However, the modulation of those functional molecules has not yet been studied, although the modulatory role of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling cascade on nicotinic acetylcholine receptor current has been reported (Courjaret and Lapied 2001). Here we describe that KNa channel is an important target molecule for OA and DA and cAMP/PKA and cyclic guanosine 3',5'-monophosphate/protein kinase G (cGMP/PKG) signal cascades are also involved in the modulation of KNa channels. |
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METHODS |
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Experiments were carried out on adult male crickets, Gryllus bimaculatus, maintained in a colony in the Department of Biology at 25–30°C with a relative humidity of 65–85% under a 12-h/12-h light/dark photoperiod. Crickets were fed on an artificial insect diet (Oriental Yeast) and supplied with water. The present experiments were performed under the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, recommended by the Physiological Society of Japan.
Kenyon cell isolation
Adult male crickets were anesthetized using CO2 before dissection. The brain was carefully removed from the head capsule, taking care not to tear the alimentary tract. The mushroom bodies were then dissected out of the brain and placed in a silicone chamber (volume of 3 ml) filled with Ca2+-free normal saline and incubated for 15 min. The mushroom bodies were then transferred to the vial tube containing dissociation solution (Sumitomo nerve-cell culture medium, Sumitomo Bakelite). The isolated mushroom bodies were incubated in this culture medium for 30 min at 25°C. After incubation, the pooled mushroom bodies were rinsed with normal saline and dissociated by gentle trituration through a fire-polished pipette with an inner diameter of about 100 µm.
Patch-clamp recording
Patch pipettes were pulled from capillary tubes (G-1.5, Narishige) with a two-stage pipette puller (PC-10, Narishige) and had a tip resistance of about 5 M
when filled with a solution for each experiment. Freshly dispersed cells were allowed to settle on the flat-glass bottom of a silicone chamber mounted on the stage of an inverted microscope (Ix70, Olympus) and the patch electrode was positioned on the cell surface with a three-dimensional hydraulic micromanipulator (MHW-3, Narishige). Single-channel currents were recorded in cell-attached patch configuration, as described in Hamill et al. (1981), through a patch-clamp amplifier (Axopatch 200B). Some experiments were performed in the inside-out configuration by pulling the electrodes from the cell-attached membranes. Current signals were sampled at 5 kHz and were low-pass filtered at 1 kHz (six-pole Bessel). Digitized signals were further analyzed by personal computer using pClamp 9.2 software (Axon Instruments). All experiments were performed at room temperature (20–25°C).
Analysis of single-channel currents
Amplitudes of single-channel currents were measured by eye or a cursor on Clampfit at different holding potentials and the single-channel conductance was measured from the slope of the current–voltage (I–V) relationship. Membrane potentials and reversal potentials are defined as the potential at the cytosolic face of membrane with respect to the potential at the external face of the membrane. Multiple channels were present in every membrane patch. As a consequence, open probability (Po) was expressed as NPo, where N represents the number of single channels, and calculated using the following expression: NPo = (A1 + 2A2 + 3A3 + ... + nAn)/(A0 + A1 + A2 + ... + An), where A0 is the area under the curve of an all-point amplitude histogram corresponding to the current in the closed state, and A1 – An represents the histogram areas reflecting the different open-state current levels for 1 – n channels present in the patch. Histogram parameters were obtained from multiple least-squares Gaussian fits of the data using Clampfit 9.2 software. The single KNa channel currents recorded from the cell-attached patch membranes often showed subconductance levels of various amplitudes between the closed and the full-open levels. Therefore we focused only on the KNa channel activity with full-open level. Averaged data are expressed as the means ± SE, where n equals the number of patches (cells).
Backfill procedure
In some experiments, Cl– channel blockers [niflumic acid (50 µM), 9-anthracenecarboxylic acid (9-AC, 50 µM), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 100 µM)] and K+ channel blockers [tetraethylammonium chloride (TEA, 1 or 10 mM) and iberiotoxin (100 nM)] were applied from the outside of the patch membranes by using the backfill procedure. Briefly, tips of freshly pulled pipettes were filled by being placed in a 1-ml sample vial containing a filtered solution (140 mM K+) for 2–3 min and then the shanks were backfilled with solutions containing those chemicals. In this experiment, the effects of these chemicals became visible 3–5 min after the backfill. Therefore we measured the effect of these chemicals on a single channel 5 min after the backfill.
Solution and chemicals
Patch-clamp recordings of single K+ channel activity were made from cell-attached patches by using pipettes filled with (in mM): 140 KCl and 5 HEPES, buffered to pH 7.4 (Tris). The cell resting potential was zeroed with an external solution containing (in mM): 140 KCl, 10 NaCl, 44 glucose, and 2 HEPES, buffered to pH 7.4 (Tris). In excised inside-out patch recordings, the pipette solution contained (in mM): 140 KCl and 5 HEPES, buffered to pH 7.4 (Tris) and the bath solution contained (in mM): 140 KCl, 10 NaCl, 44 glucose, and 2 HEPES, buffered to pH 7.4 (Tris). In some experiments, extracellular NaCl (10 mM) was replaced with NaCl (0 mM), NaCl (5 mM), NaCl (30 mM), and LiCl (30 mM). Niflumic acid, 9-AC, DIDS, KT5823, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), and quinidine were dissolved in dimethylsulfoxide to make stock solutions.
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RESULTS |
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Single-channel currents were recorded in cell-attached patch with high K+ (140 mM) in the patch pipettes at both positive and negative membrane potentials (Fig. 1, A and B). At membrane potentials of +50 and +60 mV, the unitary currents in the outward direction were recorded and they displayed slower open–closed kinetics. On the other hand, the unitary currents in the inward direction were recorded and they displayed burst-like activity with short open times interrupted by very brief closed times at membrane potentials of –50 and –60 mV (Fig. 1B). The amplitudes of single-channel currents were calculated from all-point amplitude histograms at each holding potential and the I–V relationship was obtained by plotting the current amplitude against the membrane potentials (Fig. 1C). The average slope conductance was calculated to be 127 ± 6 pS (n = 4–6) in the potential range –40 to +60 mV.
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To examine the selectivity of this channel for K+, single-channel currents were measured under different external K+ concentrations ([K+]o) and the I–V relationship was obtained. With 70 mM [K+]o, the slope conductance was 122 ± 5 pS (n = 4–6) in the potential range –70 to +60 mV and the reversal potential obtained by extrapolating the regression line was –15 ± 2 mV (Fig. 2, A1 and A2). With 20 mM [K+]o, the slope conductance was 84 ± 7 pS (n = 4–5) in the potential range –30 to +60 mV and the reversal potential was –40 ± 3 mV (Fig. 2, A2 and B2). The theoretical values of the reversal potential under these conditions were calculated by using the Nernst equation, assuming that [K+]i is 140 mM. The formula is: E = RT/zF ln ([K+]o/[K+]i), where R is the universal gas constant, T is the absolute temperature, F is the Faraday (electric charge per gram equivalent of univalent ions), z is the atomic number, [K+]o and [K+]i are the potassium concentrations on each side, and ln is the natural logarithm. The calculated values of reversal potential are as follows: 0 mV at 140 mM [K+]o; –17 mV at 70 mM [K+]o; and –49 mV at 20 mM [K+]o. These values are close to those obtained by extrapolation of the regression lines at each [K+]o, indicating a high selectively of this channel for K+.
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To determine whether the unitary current activities recorded in cell-attached patch configuration are K+ currents passing through Na+-activated K+ channels, single-channel currents were recorded in inside-out patch configuration. After excising the membrane into the inside-out configuration, the dependence of K+ channel activation on internal Na+ concentration was examined under the conditions with the same ionic strength and with the same [K+]o (120 mM) (Fig. 3, A1, A2, and A3). When the cell-attached recordings had been obtained, the patches were excised and transferred into bath solution containing high Na+. As shown in Fig. 3A, there was little or no channel activity in a nominally Na+-free bathing solution (120 mM KCl, 30 mM choline chloride). When the bathing solution was replaced with a solution containing high Na+ (5 mM NaCl, 120 mM KCl, 25 mM choline chloride), channel activity increased (Fig. 3A2). Further increase in NaCl (30 mM NaCl, 120 mM choline chloride) in bath solution greatly enhanced the Po of the single-channel currents (Fig. 3A3). The single-channel conductance, however, remained unchanged; the average slope conductance was 130 ± 8 pS (n = 8–10) with 5 mM NaCl (Fig. 3B1) and 143 ± 7 pS (n = 12–15) with 30 mM NaCl (Fig. 3B2).
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It has been suggested that KNa channels are inhibited by intracellular Li+ (Bishoff et al. 1998). Therefore we investigated the effects of the replacement of intracellular Na+ with an equimolar Li+. Records were obtained from an inside-out patch membrane in a solution containing high Na+ (30 mM NaCl, 120 mM KCl) showing high activities (Fig. 4A1). When all intracellular Na+ was replaced by Li+, the single-channel current activities were drastically reduced (Fig. 4A2). The Po of the KNa channel was 0.693 with 30 mM Na+ and 0.131 with 30 mM Li+. Average values of Po examined from seven different cells were as follows: 0.66 (control) and 0.09 (30 mM Li+). Next we examined the action of quinidine, which is widely used as a KNa channel blocker. Figure 4B1 shows the control record showing unitary activities of KNa channel under the inside-out patch configuration. Intracellular Na+ was 30 mM and the membrane potential was set to +30 mV. When 100 µM quinidine was applied to the bath solution, the Po of KNa channel currents decreased by 54 ± 13% (n = 8) (Fig. 4B3), indicating that the KNa channel in cricket Kenyon cells is quinidine sensitive.
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Figure 5A shows single KNa channel activity obtained from the inside-out patch membrane in the high K+ bath solution containing 30 mM Na+. When this high K+ bath solution was replaced with a high K+ solution that contained 30 mM Na+ and 10 µM Ca2+, the Po of KNa channel was drastically reduced from 0.638 (control) to 0.102 (pCa = 5). Average values of Po examined from eight different cells were as follows: 0.65 (control) and 0.09 (pCa = 5). As a result of the decrease in Po of KNa channel current by intracellular Ca2+, it was difficult to resolve the unitary currents.
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To examine whether KNa channels are inhibited by blockers known to block K+ channels, TEA and iberiotoxin were added to the patch pipettes by the backfill procedure. Recordings of K+ channel activity were made in a cell-attached patch. As shown in Fig. 6A1 the amplitude of the single-channel currents was reduced by adding 1 mM TEA. On increasing the concentration of TEA from 1 to 10 mM, the amplitude of the single-channel currents was further reduced. The average values of relative inhibition of the single-channel current amplitude were 49 ± 14% (n = 5; 1 mM TEA) and 82 ± 11% (n = 5; 10 mM TEA) (Fig. 6A2). Next we examined the effect of iberiotoxin, known as a specific blocker for the large-conductance Ca2+-activated K+ channel on the unitary current activities (Fig. 6, B1, B2, and B3). All-point amplitude histograms obtained in the absence and presence of iberiotoxin (100 nM; shown in the bottom of each current trace) clearly showed that there is no obvious change in either the amplitude or the Po of single-channel currents.
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We investigated the effect of OA and DA on the unitary current activities of the KNa channel under the cell-attached patch configuration. Figure 7A1 shows control records in the absence of OA at a holding potential of +50 mV. When 1 µM OA was applied to the bath solution, the channel activities gradually increased (Fig. 7, A2 and A3). As a result, the Po of the KNa channel increased from 0.088 (control) to 0.374 (210 s after) and 0.713 (295 s after) (Fig. 7, B1 and B2). The average values of Po examined from six different cells were as follows: 0.11 (control), 0.39 (200 s after), and 0.68 (300 s after). Next we examined the effect of DA on the unitary current activities of the KNa channel. Figure 7B1 shows control records in the absence of DA, illustrating the activity of the unitary currents at a holding potential of +30 mV. When 1 µM DA was applied to the bath solution, the Po of the KNa channel currents decreased from 0.389 (control) to 0.158 (1 µM DA). The average values of Po examined from six different cells were as follows: 0.47 (control) and 0.20 (1 µM DA).
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To investigate whether the cAMP and cGMP signaling pathways are involved in the modulation of KNa channel, we examined the effect of 8-bromoguanosine-3',5'-cyclic adenosine (8-Br-cAMP) and 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP) on the KNa channel currents. Figure 8A1 shows control records illustrating the activity of the unitary currents at +50 mV. When 1 mM 8-Br-cAMP was applied to the bath solution, the Po of KNa channel increased drastically from 0.113 to 0.586 (Fig. 8A2). Next to examine whether the downstream pathway of cAMP involves PKA activation, we investigated the effects of the PKA inhibitor H-89. As shown Fig. 8A3, the addition of H-89 (1 µM) reduced the Po from 0.586 to 0.155, indicating that the excitatory action of cAMP on the KNa channel is via phosphorylation by PKA. The average values of Po examined from eight different cells were as follows: 0.13 (control), 0.61 (1 mM 8-Br-cAMP), and 0.16 (1 mM 8-Br-cAMP plus H-89). Next we examined the effect of 8-Br-cGMP on the unitary currents. Figure 8B1 shows control records in the absence of 8-Br-cGMP at a holding potential of +40 mV. When 1 mM 8-Br-cGMP was applied to the bath solution, the Po of KNa channel drastically decreased from 0.483 to 0.088 (Fig. 8B2). To determine whether the inhibition by cGMP involves PKG activation, we investigated the effect of the PKG inhibitor KT5823. As shown in Fig. 8B3, the addition of KT5823 (1 µM) increased the Po from 0.09 to 0.42, indicating that the inhibitory action of cGMP on KNa channels is via phosphorylation by PKG. The average values of Po examined from five different cells were as follows: 0.55 (control), 0.11 (1 mM 8-Br-cGMP), and 0.51 (1 mM 8-Br-cGMP plus KT5823).
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DISCUSSION |
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KNa channels in cricket Kenyon cells
The results presented here provided strong evidence for the presence of KNa channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. They possess a large conductance of 143 ± 7 pS in 140 mM [K]o/140 mM [K]i solutions and show little voltage dependence. It was also found that for activation of the channel, intracellular Na+ is necessary, although Li+ could not substitute for Na+ in activation of KNa channels. The KNa channel currents activated by intracellular Na+ were first detected in mammalian cardiac cells (Kameyama et al. 1984) and in crayfish neuron (Hartung 1985). Subsequently, similar channels were reported in a variety of neuronal cells (Bader et al. 1985; Bischoff et al. 1998; Dale 1993; Dryer 1991; Dryer et al. 1989; Egan et al. 1992; Haimann et al. 1992; Safronov and Vogel 1996; Schwindt et al. 1989). KNa channels are also found in cockroach DUM neurons (Grolleau and Lapied 1994) and in cultured Drosophila neurons (Saito and Wu 1991).
In cardiac myocytes, the KNa channel has an EC50 for Na+ of 66 mM with Hill coefficient of about 3 (Kameyama et al. 1984). In brain stem neurons, the KNa channel can be activated by 20 mM intracellular Na+ and shows no sign of saturation even at 150 mM (Dryer 1991; Dryer et al. 1989). In trigeminal ganglion cells, the KNa channel has an EC50 for Na+ of 27–29 mM with Hill coefficient of about 2 (Haimann et al. 1990). Therefore the KNa channels so far described seem to require a high concentration of Na+ for activation of the channel. However, KNa channels described here seem to be highly sensitive to Na+ because even low concentrations of Na+ (5 mM) could activate KNa channel currents, although the EC50 value for Na+ was not determined in the present preparation.
It has been shown that almost all neuronal KNa channels so far described are insensitive to intracellular Li+ (Bishoff et al. 1998; Dryer et al. 1989; Dryer 1991; Haimann et al. 1990; Safronov and Vogel 1996). On the other hand, KNa channels in crayfish motoneurons reported by Hartung (1985) were activated by intracellular Li+ as well as Na+. The present results showed that the replacement of intracellular Na+ with Li+ drastically reduced the Po of the channel currents but the currents still appeared. These results indicate that Li+ is actually an activator of the channel but a much less effective one than Na+. It has also been reported that vertebrate KNa channels are blocked by intracellular Ca2+ (Dryer et al. 1989; Haimann et al. 1990, 1992). Similarly, the KNa channel in cricket Kenyon cells is also found to be blocked by intracellular Ca2+, indicating that a common intracellular mechanism exists between the vertebrate and insect KNa channel.
It has been revealed that the single-channel conductance of the KNa channel is strongly dependent on [K+]o and [K+]i (Dryer et al. 1989; Mistry et al. 1997; Safronov and Vogel 1996). Similarly, the conductance of the KNa channel in cricket Kenyon cells changes its value depending on the [K+]o/[K+]i; when [K+]o was increased from 20 to 140 mM, the slope conductance increased from 84 ± 7 pS (n = 4–5) to 127 ± 6 pS (n = 4–6). The conductance value of 127 pS with a high K symmetrical condition found in the present preparation is closely similar to the values obtained from that of vertebrate. Thus the properties of KNa channels in cricket Kenyon cells are quite similar to those reported in many other vertebrates with respect to their conductance value, [K+]o/[K+]i dependence of single-channel conductance, and Li+ and Ca2+ sensitivities. Our results showed that the KNa channel current activities were most frequently recorded by the cell-attached membranes. This aspect is consistent with the notion reported by Grünewald (2003) that the main component of macroscopic outward current is not Ca2+ activated.
Inhibition by TEA and quinidine
The present results showed that TEA blocks KNa channels even at low concentration (1 mM), whereas iberiotoxin had little effect even at high concentration (100 nM) in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. The recent molecular biological studies on KNa channels have revealed that there are two types of genes that encode KNa channels: the Slack (Slo2.2) and Slick (Slo2.1) genes (Bhattacharjee et al. 2002; Joiner et al. 1998; Yuan et al. 2003). Slick has been shown to be expressed in the nervous system and heart, whereas Slack has selectively been expressed in the nervous system. Single-channel conductance is similar between them and both channels show outward rectification and are activated by intracellular Na+ (Bhattacharjee et al. 2003). In both the Slick and Slack channels, TEA is effective from the outside (Bhattacharjee et al. 2003), whereas 100 nM iberiotoxin is less effective (Bhattacharjee et al. 2003). In this respect, the KNa channel identified in the cricket Kenyon cells is basically similar to Slick and Slack channels except for the property of outward rectification.
One of the noteworthy properties of the KNa channels in cricket Kenyon cells is its blockade by quinidine. Quinidine has been shown to inhibit the delayed rectifier K+ current (IK; Balser et al. 1991; Furukawa et al. 1989; Hiraoka et al. 1986; Roden et al. 1988), the inward rectifier K+ current (IKI; Balser et al. 1991; Hiraoka et al. 1986; Salata and Wasserstrom 1988), and the transient outward current (Imaizumi and Giles 1987). In the single ventricular cells of guinea pig heart, quinidine inhibited the KNa channel current by decreasing the open time by direct binding to the channel (Mori et al. 1998). Quinidine has also been shown to inhibit both Slick and Slack currents at a concentration of 1 mM (Bhattacharjee et al. 2003). In honeybee Kenyon cells, quinidine-sensitive outward currents were also reported with the use of whole cell patch-clamp technique (Schäfer et al. 1994). Therefore it seems possible that the quinidine-sensitive KNa channel may underlie the major component of whole cell outward current in Kenyon cells.
Involvement of cAMP and cGMP signaling pathway
Santi et al. (2006) clarified that KNa channels are important target molecules for neuromodulators through G
q-protein–coupled receptors (GqPCRs) and implicated their important role in the effect of long-lasting changes on neuronal excitability. In this study, we have found for the first time that the KNa channel in cricket Kenyon cells is an important target molecule for monoamines, OA and DA. Previous studies on honey bees (Hammer and Menzel 1998; Kreissl et al. 1994; Menzel et al. 2001) and fruit flies Drosophila (Schwaerzal et al. 2003) suggest that acquisition with sugar depends on the octopaminergic system, whereas acquisition with electric shock depends on the dopaminergic system. The VUM neuron originated in the subesophageal ganglion has been shown to be octopaminergic and it carries the appetitive (sucrose) signals (Menzel et al. 1999). Direct injection of OA in termination areas of this neuron could substitute presentation of a sucrose reward in olfactory condition (Hammer and Menzel 1998). In cricket, it is also suggested that OA and DA act as neurotransmitters that convey the reward and punishment signals (Unoki et al. 2005). A molecular biological study using RNA interference of OA receptors further confirms this aspect (Farooqui et al. 2003). In many species, including mollusca Aplysia, fruit flies Drosophila, and mice, suggest that formation of long-term memory requires an increase in intracellular cAMP and recruitment of the PKA that phosphorylates the transcription factor, cAMP-responsive element-binding protein, CREB (Abel et al. 1998; Bartsch et al. 1995; Yin et al. 1995). Participation of mushroom bodies in learning and memory is considered to involve the cAMP signaling pathway because the affected genes of three Drosophila mutants—dance, rutabaga, and DCO—all defective in learning, encode for the enzymes cAMP phosphodiesterase, adenylate cyclase, and protein kinase A (Davis 1996; Han et al. 1992; Nighorn et al. 1991; Skoulakis et al. 1993). The importance of cGMP in various physiological functions in brain are also implicated (Hofmann et al. 2000; Schmidt and Walter 1994). The most extensively studied cGMP signal transduction pathway is that triggered by nitric oxide (NO) (Bredt and Snyder 1990). Matsumoto et al. (2006) have shown that the NO–cGMP pathway stimulates the cAMP pathway to induce long-term memory (LTM) in the cricket Gryllus bimaculatus. They have implicated that the NO–cGMP pathway activates the adenylyl cyclase (AC)–cAMP–PKA–CREB signaling pathway via cyclic nucleotide-gated channel and Ca2+–CaM and thereby results in protein-synthesis–dependent LTM. The present studies show that 8-Br-cAMP increased the Po of KNa channel currents, whereas 8-Br-cGMP decreased the Po of KNa channel currents. It has to be determined whether OA and DA receptor activation triggers the signal cascade of cAMP/PKA and cGMP/PKG, respectively, or whether DA receptor activation triggers the signal cascade that leads to inhibition of AC.
Physiological significance of the modulation of KNa channel
A physiological role for KNa channels identified in Kenyon cells isolated from the cricket mushroom bodies is not yet clear. In other neuronal cells, it has been proposed that Na+ influx through voltage-gated Na+ channels during an action potential may produce a transient activation of KNa channels, resulting in action potential repolarization (Bader et al. 1985; Dryer et al. 1989). The present studies have revealed that Po of KNa channels is increased and decreased by OA and DA, respectively. Therefore it can be considered that increased Po by OA may result in a shortening of Na+-dependent action potential duration, whereas DA results in a prolongation of action potential duration. Further studies are still necessary to clarify whether the possible change of action potential shape occurs when we associate CS and US in Kenyon cells.
In conclusion, we have shown that OA, which mediates reward information, increases the Po of KNa channel, whereas DA, which mediates a punishment signal, decreases the Po of KNa channel in cricket Kenyon cells. cAMP/PKA and cGMP/PKG signaling pathways are also found to be involved in the modulation of KNa channel, probably via phosphorylation of target protein (KNa channel) (Fig. 9).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Yoshino, Department of Biology, Tokyo Gakugei University, Tokyo 184-8501, Japan (E-mail: myoshi{at}u-gakugei.ac.jp)
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INTRODUCTION
