Article Figures & Data
Figures
- Figure 1.
Calcium responses in isolated honeybee KCs during temporal pairing of ACh and GABA. A, Isolated honeybee KCs 24 h after preparation. B, Stimulation scheme. In each experiment isolated KCs received a short pulse of ACh (3 s) as the first stimulus. The second stimulus was ACh (3 s), ACh—GABA, or GABA–ACh (3 s each) followed by the third stimulus, which was ACh (3 s) for all groups. C, Pairing of ACh with GABA significantly inhibited calcium responses during the second stimulation. After ACh–GABA pairing, peak response of the third stimulation was increased while a GABA–ACh pairing led to elevated calcium levels during the late phase (shaded area). Shaded areas around peaks indicate time windows used for correlation analysis in D. The data derived from seven independent measurements (ACh: n = 110 neurons; ACh–GABA: n = 83 neurons; GABA–ACh: n = 107 neurons). Two-way ANOVA followed by Bonferroni post hoc test: a, ACh vs ACh–GABA, p < 0.001; b, ACh vs GABA–ACh; p < 0.008. D, For ACh and ACh–GABA peak responses during second and third stimulation showed strong correlation. No such correlation was observed for GABA–ACh pairing demonstrating substantial pairing-specific differences. Statistics used was the Pearson correlation. E, The decay time in the late phase after ACh–GABA and GABA–ACh pairing is significantly slower compared with ACh alone. During the third stimulation only the GABA–ACh pairing led to a slower decay time, which resulted in elevated calcium levels during the late phase. Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test, *p < 0.05.
- Figure 2.
Specificity and concentration dependence of pairing-related inhibition and plasticity. A, Inhibition during ACh–GABA pairing was blocked by the chloride channel blocker PTX. Data derived from two independent measurements for each group (ACh–GABA, 5 μm PTX: n = 22; ACh–GABA, 10 μm PTX: n = 28; ACh–GABA, 50 μm PTX: n = 29; ACh, 50 μm PTX: n = 36). Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test, *p < 0.05. B, Elevation of GABA concentration during GABA–ACh pairing increases inhibition. Data derived from at least four independent measurements for each group (ACh: n = 110 neurons; GABA–ACh, 1:1: n = 107 neurons; GABA–ACh, 2:1: n = 39 neurons). Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test, *p < 0.05. C, Presentation of GABA alone has no effect on subsequent third stimulation. Data derived from four independent measurements (ACh: n = 37 neurons; GABA: n = 35 neurons). Two-way ANOVA followed by Bonferroni post hoc test: p > 0.15.
- Figure 3.
ACh-mediated calcium responses of honeybee KCs are modulated by ionotropic and metabotropic GABA receptors. Inhibition of ACh-induced calcium influx during second stimulation was mediated by ionotropic GABA receptors (selective agonist muscimol). Both, ionotropic and metabotropic (3-APMPA) GABA receptors contributed in facilitating the ACh-induced calcium influx during the third stimulation. Data derived from at least seven independent measurements (ACh: n = 150 neurons; ACh–3-APMPA: n = 141 neurons; ACh–muscimol: n = 147 neurons). Two-way ANOVA followed by Bonferroni post hoc test: a, ACh vs ACh–Muscimol; b, ACh vs ACh–3-APMPA; p < 0.002.
- Figure 4.
Calcium responses in a defined subset of isolated Drosophila KCs during temporal pairing of ACh and GABA. A, Fluorescence image of larval brain whole mount (201y). The calcium-sensitive fluorescence protein cameleon is specifically expressed in a subset of MB KCs involved in learning. B, The isolated KCs were identified by their characteristic cameleon fluorescence (arrows). C, Pairing of ACh and GABA during the second stimulation significantly inhibited the calcium response. ACh–GABA pairing led to elevated calcium levels during the late phase of the third stimulation. Shaded areas indicate averaged time windows used in D. Data derived from at least five independent preparations (ACh: n = 22 neurons; ACh–GABA: n = 17 neurons; GABA–ACh: n = 22 neurons). Two-way ANOVA followed by Bonferroni post hoc test: a, ACh vs ACh–GABA; b, ACh vs GABA–ACh; p < 0.02. D, Calcium responses for single KCs showed substantial pairing-specific differences and division into subpopulations with different properties. E, F, Strongly inhibited KCs showed no calcium response during the second stimulation and elevated calcium levels during the late phase of the third stimulation. The other subpopulation of KCs was only weakly inhibited and showed no difference during the third stimulation. Two-way ANOVA followed by Bonferroni post hoc test: b, ACh vs GABA–ACh; p < 0.02.
