Inhibition of mAChR does not impair the reduction of Ca2+ responses in the MB calyx
Using a mAChR antagonist, atropine (Collin et al., 2013), I examined whether mAChR also contributes to the reduction of Ca2+ responses in the MB calyx. After AL stimulation with 40 stimulus trains, Ca2+
responses in the calyx were reduced regardless of the presence or absence of atropine (Fig. 24). Thus, it is unlikely that mAChR is a key molecule that induces the reduction of Ca2+ responses in the calyx.
The activation of nAChRs increases the intracellular cAMP level and [Ca2+]i in the MB calyx First, I determined whether nAChRs activation induce cAMP production in the calyx. I observed responses of cAMP and Ca2+ in the calyx simultaneously, using Epac1-camps and the red-shifted Ca2+
indicator R-GECO1 (Zhao et al., 2011). Since ACh is a major neurotransmitter in the Drosophila brain, the dissected brain was treated with TTX to prevent the generation of action potentials and
non-cell-autonomous responses. Intracellular cAMP level and [Ca2+]i increased following an agonist of nAChRs, nicotine application in the calyx (Fig. 25) in a dose-dependent manner (Fig. 26). Next, to confirm the nicotine-induced cAMP and Ca2+ responses are nAChRs-dependent, I treated the nAChR antagonist, mecamylamine (Kazama & Wilson, 2008) before nicotine application. Both cAMP and Ca2+
responses were completely suppressed in the presence of mecamylamine (Fig. 26). Thus, these results indicate that the stimulation of nAChR increases cAMP level in the MB calyx.
Ca2+ influx through nAChRs produces cAMP in the MB calyx
Since some types of ACs produce cAMP by Ca2+, I hypothesized that nicotine-induced cAMP production is due to the increase of [Ca2+]i. To examine whether cAMP production depends on [Ca2+]i,
nicotine-induced responses were observed under Ca2+ free conditions using pretreatment of the membrane-permeable Ca2+ chelator BAPTA-AM before nicotine application. The treatment of BAPTA-AM completely abolished Ca2+ responses in the MB calyx (Figs. 27B and D). Furthermore,
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BAPTA-AM pretreatment suppressed cAMP production induced by nicotine compared with the control (Figs. 27A and C).
The stimulation of nAChRs induces Ca2+ influx from extracellular solution and Ca2+ efflux from intracellular Ca2+ stores in KCs (Campusano et al., 2007). Next, I changed external Ca2+ concentration to examine whether Ca2+ influx from extracellular solution induces cAMP production. Nicotine-inducible cAMP responses in a high-external-Ca2+-concentration solution (3.6 mM) were significantly stronger than those in the normal solution (Fig. 28; 3.6 mM Ca2+ vs. 1.8 mM Ca2+). In contrast, cAMP production was suppressed in the Ca2+-free external solution (Fig. 28; Ca2+ free vs. 1.8 mM Ca2+). Thus, cAMP
production in the calyx mainly depends on the Ca2+ influx induced by nicotine.
The activation of nAChRs induces Rut-AC-dependent cAMP production in the MB calyx
Since Rut-AC is Ca2+/calmodulin-responsive AC (Levin et al., 1992), I observed nicotine-induced cAMP responses in the calyx of rut1 mutant. The nicotine-induced cAMP responses in the normal solution in rut1 were weaker than that in the wild-type [Figs. 29A and C; rut1 (1.8 mM Ca2+) vs. WT (1.8 mM Ca2+)].
However, the Ca2+ responses were also suppressed in rut1 compared with wild-type [Figs. 29B and D; rut1 (1.8 mM Ca2+) vs. WT (1.8 mM Ca2+)]. In the previous study also reported that nicotine-induced Ca2+
responses were suppressed in the MB by rut mutations (Pavot et al., 2015). In this situation, it is possible that the decrease of Ca2+ influx by rut mutation suppress cAMP responses. Thus, to exclude the
possibility I applied nicotine in the low-external-Ca2+-concentration solution. The Ca2+ response in the wild-type in the low-external-Ca2+-concentration solution was similar to in rut1 in the normal solution [Figs. 29B and D; rut1 (1.8 mM Ca2+) vs. WT (0.9 mM Ca2+)]. In contrast, the cAMP responses in the normal solution in rut1 were weaker than that in the low-external-Ca2+-concentration solution in the wild-type [Figs. 29A and C; rut1 (1.8 mM Ca2+) vs. WT (0.9 mM Ca2+)]. These results support the idea that Rut-AC actually produces cAMP in the calyx in a Ca2+-dependent manner.
Finally, I stimulated ACs directly using forskolin to determine whether cAMP production by the activation of ACs can induce Ca2+ influx into the calyx. Forskolin treatment did not induce Ca2+ influx
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into the calyx (Figs. 30B and D), whereas it increased cAMP level with or without extracellular Ca2+
(Figs. 30A and C), indicating that cAMP production alone is not sufficient to induce Ca2+ influx into the calyx. Taken together, it is most likely that Rut-AC-dependent cAMP production in the calyx results from the elevation of [Ca2+]i induced by nAChRs activation.
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Discussion
In this study, I revealed nAChRs-induced cAMP production in the MB calyx. The application of nicotine increased [Ca2+]i and cAMP level, and the responses were completely suppressed using nAChR blocker (mecamylamine) in the MB calyx (Fig. 26). Nicotine-induced cAMP production was suppressed under external Ca2+ free conditions (Fig. 28). Conversely, the cAMP production was enhanced in a
high-external-Ca2+-concentration solution (Fig. 28). Thus, nAChRs-induced cAMP production depends on Ca2+ influx. Furthermore, the nicotine-induced cAMP response was also suppressed in rut1 mutant flies (Fig. 29). In contrast, an AC activator, forskolin-induced cAMP production was not changed under external Ca2+ free conditions (Fig. 30). Furthermore, forskolin treatment did not increase an elevation of [Ca2+]i (Fig. 30). These observations suggested that cAMP production is downstream of Ca2+ influx.
Taken together, it is possible to explain nAChRs-induced cAMP production in the MB calyx as follows.
In wild-type, Ca2+ influx through nAChRs stimulates Rut-AC, and Rut-AC produces cAMP (Fig. 31A).
However, in rut1 mutant, Rut-AC dependent cAMP is not produced, while Ca2+ influx occurs via nAChRs (Fig. 31B). In external Ca2+ free conditions, the activation of nAChRs cannot increase [Ca2+]i, and
Rut-AC-dependent cAMP production was not also induced (Fig. 31C). Since forskolin directly stimulates Rut-AC, forskolin-induced cAMP production does not depend on extracellular Ca2+ concentration, and forskolin treatment does not change [Ca2+]i (Fig. 31D).
Although my study using nicotine revealed that cAMP production depends on [Ca2+]i in the calyx, slight cAMP production was detected under Ca2+-free conditions (Figs. 27 and 28). However, blocking nAChRs with mecamylamine completely suppressed the cAMP production in the calyx (Fig. 26), suggesting that nAChRs contribute to the slight production of cAMP under Ca2+-free conditions. nAChRs are widely expressed in the fly brain (Fayyazuddin et al., 2006), and nicotine treatment stimulates many neurons in the brain. Thus, nicotine treatment may induce non-cell autonomous responses in the calyx.
Because TTX and Ca2+ chelators were used in the experiments, it is most unlikely that the non-cell autonomous responses are caused by synaptic transmission. In mammalian sensory neurons, there are neuropeptides that are secreted by exocytosis of dense-core vesicles in a Ca2+-independent but
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voltage-dependent manner (Zhang & Zhou, 2002; Chai et al., 2017). Similarly, in the Drosophila brain, neuropeptides released following nicotine administration may diffuse through a large area in the brain and induce cAMP production in the calyx. Various neuropeptide receptors are expressed in MB neurons (Crocker et al., 2016), and some of them are known to promote cAMP production such as those encoded by the Pigment-dispersing factor receptor (Hyun et al., 2005; Mertens et al., 2005), Diuretic hormone 44 receptor 1 (Johnson et al., 2004), and Diuretic hormone 44 receptor 2 (Hector et al., 2009). These reports support my idea that nicotine treatment may induce neuropeptide secretion, which subsequently induces cAMP production in the calyx under Ca2+-free conditions.
In rut1 mutant, nicotine-evoked Ca2+ response was decreased in the calyx (Fig. 29). The in vivo Ca2+ imaging study showed that nicotine-induced Ca2+ responses in the calyx are suppressed in rut mutant flies (Pavot et al., 2015). Furthermore, the in vitro Ca2+ imaging study reported that nicotine-evoked Ca2+
responses in KCs are decreased by nicotine pre-exposure, and the plasticity requires Ca2+ influx and AC activity (Campusano et al., 2007). Taken together, I suggest that Ca2+-stimulated Rut-AC contributes to attenuation of nAChRs-dependent Ca2+ responses in the MB calyx.
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Figure 24. mAChR inhibition does not impair the reduction of Ca2+ responses
Ca2+ responses were recorded in the α lobe and calyx in the same brain. The reduction of Ca2+ responses was not suppressed by the treatment of the mAChR blocker atropine (red line; N = 8) compared with that of saline as the control (black line; N = 8) in the wild-type (+; +; MB-LexA LexAop-GCaMP6m/+).
Asterisks indicate the statistical significance of the difference between before and after the application of 40 stimulus trains. The hash symbols indicate the statistical significance of the difference between atropine treatment and saline treatment. Student’s t-test or the Mann–Whitney U test was used for
statistical analysis.Error bars represent SEM. NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001;
# P < 0.05; ## P < 0.01. (A) The upper panel shows typical Ca2+ responses in the calyx 3 min before (Pre) and 12 min after the application of 40 stimulus trains. (B) The upper panel shows typical Ca2+ responses in the α lobe 6 min before (Pre), during, and 9 min after the application of 40 stimulus trains.
A B
Pre
1 sec. 10 %
12 min.
1 sec. 10 %
40 trains
10 sec. 50 %
Pre
1 sec. 10 %
9 min.
1 sec. 10 %
Calyx α lobe
0 0.2 0.4 0.6 0.8 1 1.2
-10 0 10 20 30
0 0.2 0.4 0.6 0.8 1 1.2
-10 0 10 20 30
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R ela tiv e Ca
2+res pons es
Time (min.) 40 trains
Saline
100 μM atropine
Calyx R ela tiv e Ca
2+res pons es
Time (min.) 40 trains
Saline
100 μM atropine
α lobe
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NS
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*
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*
# #
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Chapter 2
Results
Figure 3
Atropine calyx and a lobe
62 Figure 25. Typical cAMP and Ca2+ responses in the calyx
Typical Epac1-camps and R-GECO1 responses in the wild-type calyx before and after 1 μM nicotine application. +; MB-LexA LexAop-R-GECO1/+; 30Y UAS-Epac1-camps/+ male was used. (A) The upper pictures show a cAMP response. Pseudo-color represents the ratio of CFP/YFP fluorescence intensity.
The bottom pictures show a Ca2+ response. Pseudo-color represents the intensity of R-GECO1 fluorescence. The area enclosed by a dotted circle is the calyx. (B) Time course of cAMP and Ca2+
responses in the MB calyx. The black line indicates cAMP response and the red line indicates a Ca2+
response. The black rectangle indicates the time of nicotine application.