Optimizing the training protocol.

<p>In (A-C), the paired color presentation preceded the electric shock application by 4 s. (inter-stimulus-interval (ISI) = -4s). (A) To find the best parameters for visual conditioning we varied the number of training trials, however each experimental group received the same total number of electric shock pulses (12) and the same total duration of color presentation (60 s per color). Visual learning scores depended on such variation in protocol (Kruskal-Wallis test, H = 15.02, d.f. = 4, p < 0.005). Significant difference in scores was found when comparing 4 trial- <i>vs</i>. 12 trial-training (<i>post-hoc</i> pairwise comparison, p < 0.05). Flies showed significant scores after 1, 2, 4 and 6 trials (one sample <i>t</i>-tests, T > 4.7, p < 0.001). Applying 12 trials with 5 s of color presentation and 1 electric shock pulse did not reveal significant conditioned avoidance (one sample <i>t</i>-test, T = 1.99, p > 0.1), <i>n</i> = 15–20. (B) Using the optimal conditions from (A) (dashed box), application of one to eight training trials led to significant conditioned avoidance (one-sample Wilcoxon signed rank tests, p < 0.001). Significant difference in scores was found between conditioning with one trial <i>vs</i>. 8 trials (Kruskal Wallis test, <i>post-hoc</i> pairwise comparison p < 0.05), <i>n</i> = 16 (C) Using the optimal conditions from (A) and (B) (dashed boxes), we varied the inter-color-interval (ICI) and inter-trial-interval (ITI) between 30 s and 120 s. Learning scores did not depend on the duration of the ICI or ITI (one-way ANOVA, F = 0.407, p > 0.6). All groups showed significant scores (one sample <i>t</i>-tests, T > 6.2, p < 0.001), <i>n</i> = 20. In (A-C), bars and error bars represent means and SEMs, respectively. (D) The resulting optimized training protocol with 8 training trials; each with 15 s long presentations of color stimuli, 3 electric shock pulses and an ICI and ITI of 120 s. Only one trial is sketched.</p

Effect of stimulus timing on visual memory.

<p>Conditioned behavior as a function of inter-stimulus interval (ISI). Red stripes indicate electric shock pulses. Data points indicate onset of the paired color presentation with 15 s duration (x-axis) and mean learning index (y-axis). Error bars represent the SEMs. Black data points: Learning scores depended on the ISI (one-way ANOVA, F = 13.86, p < 0.001). Flies showed significant conditioned avoidance with overlapping paired color presentation and shock pulses (one sample <i>t</i>-tests, T > 8.3, p < 0.001, ISI = -14 s, -4 s) and when the paired color preceded shock by a short temporal gap (one sample <i>t</i>-tests, T > 2.9, p < 0.01; trace conditioning, ISI = -34 s, -19 s). Flies showed significant conditioned approach when the paired color followed shock with a gap (one sample <i>t</i>-tests, T > 2.648, p < 0.05; relief conditioning, ISI = +26 s, +34 s, +49 s, +56 s, +66 s, +76 s; for ISI = +19 s and +41 s; T < 1.493; p > 0.05). When the paired color followed shock with overlap, scores did not differ from zero (one sample <i>t</i>-tests, T < 2.2, p > 0.05; ISI = +4 s, +11 s). Also when the two stimuli were too far apart in time (ISI = -64 s, -49 s, +96 s) flies showed no conditioned behavior (one sample <i>t</i>-tests, T < 1.7, p > 0.05), <i>n</i> = 12–44. Grey data points: We re-examined trace conditioning using additional negative ISIs (ISI = -34 s to -4 s). Flies showed significant conditioned avoidance with all tested ISI values (one sample <i>t</i>-tests, T > 3.0, p < 0.05). Learning scores depended on ISI value (one-way ANOVA, F = 7.355, p < 0.0001), such that the visual memory steadily decreased with increasing temporal gap (<i>post-hoc</i> pairwise comparisons p < 0.05 for -34 s <i>vs</i>. -14 s, -34 s <i>vs</i>. -4 s, -26 s <i>vs</i>. -14 s, -26 s <i>vs</i>. -4 s, -19 s <i>vs</i>. -4 s), <i>n</i> = 16–24.</p

Adenylate cyclase as a molecular coincidence detector.

<p>In a variety of associative learning systems, a potential coincidence between the trained stimulus and the reinforcement is detected at the pre-synapse by a particular kind of adenylate cyclase. The stimulus acts on the respective neurons, raising the intracellular Ca⁺⁺ concentration. The reinforcement induces the release of a transmitter that binds to its respective G protein coupled receptors (GPCR) on the very same neurons and activates the G protein (G*). If stimulus and reinforcement are appropriately timed, the two types of input act synergistically on the adenylate cyclase (AC*), triggering cAMP signalling, and thus lead to the strengthening of the output from these neurons to the respective conditioned behaviour pathway.</p

Influence of Ca⁺⁺ duration and intensity.

<p>Complementing the analysis shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g007" target="_blank">Fig. 7</a> we now vary the Ca⁺⁺ input while keeping the transmitter input fixed. In all three examples shown in (A), the Ca⁺⁺ input rises to a peak of 6·10⁻⁴ moles/L within 40 ms after the Ca⁺⁺ onset, but decays with different time constants, chosen as 0.1 s, 1 s and 10 s (A, top). In this scenario, the associative effects increase with increasing Ca⁺⁺ duration (A, bottom). In addition, a large decay constant causes a long tail of the Ca⁺⁺ input that enables negative associative effects for longer ISIs (A, the last case). In (B) we provide an exemplary Ca⁺⁺ input (B, top) which gives good fit to the behavioural results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g001" target="_blank">Fig. 1</a> in terms of the ISI-dependency of the associative effects but not in terms of their sizes relative to each other (B, bottom). In this case, the Ca⁺⁺ concentration rises to a peak of 6·10⁻⁴ moles/L within 13 s after the onset, comparing well with the 15s- long odour presentation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g001" target="_blank">Fig. 1</a>. Note that the best negative associative effect occurs with ISI = −13 s, similar to the behavioural situation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g001" target="_blank">Fig. 1</a>. Finally, in (C), we study the effects of the intensity of the Ca⁺⁺ input. We fix the transmitter input and use the Ca⁺⁺ input depicted in (B), but scaled up and down by one order of magnitude. The intensity of Ca⁺⁺ strongly influences the sizes of both the negative and the positive associative effects; the balance between the two is however somewhat compromised with increasing Ca⁺⁺ intensity.</p

Influence of the transmitter duration.

<p>With a fixed Ca⁺⁺ input, three different transmitter inputs are tested (top). They are all initiated at 210 s, rise to a peak of 7·10⁴ molecules/µm² within 40 ms after the onset, but decay with different time constants as indicated above the panels. We plot the resulting adenylate cyclase dynamics (middle) and the ISI-dependent associative effects (bottom). In terms of the percent sizes of associative effects, changing the transmitter decay time constant from 0.1 to 1 (the first two cases) hardly makes a difference. A slower decaying transmitter input (the last case) broadens the dynamics of adenylate cyclase activation/deactivation, resulting in much higher cAMP production in the control condition; thus, the percent associative effects remain small. As for the ISI-dependence of the associative effects, short transmitter inputs (the first two cases) give good fits to the situation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g001" target="_blank">Fig. 1</a>; when a slower decaying transmitter input is used (the last case), the positive associative effect only occurs for large positive ISIs, due to the broadened adenylate cyclase activation/deactivation dynamics.</p

Rate constants of the reactions for the first model.

<p>Apart from k₅ and k_-5, all values were chosen according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Rospars1" target="_blank">[46]</a>. Thus, k₁, k_-1, k₂, k_-2 were estimates from moth olfactory transduction or vertebrate phototransduction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Rospars1" target="_blank">[46]</a> for further references). For the parameters k₅ and k_-5 (see also Eqs. 13 and 14), the listed base-line values were chosen to mimic the experimentally measured dynamics of adenylate cyclase activation/deactivation in response to transmitter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Abrams3" target="_blank">[42]</a>, for a detailed sensitivity-analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g005" target="_blank">Fig. 5A</a>. k₅ and k_-5 were sensitive to Ca⁺⁺ (Eqs. 13 and 14).</p

Regulation of the adenylate cyclase by the transmitter and Ca⁺⁺.

<p>A. Adapting the model of Rospars et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Rospars1" target="_blank">[46]</a>, the transmitter reversibly binds to its respective G protein coupled receptor (GPCR) to form a complex, resulting in reversible receptor activation (GPCR*). GPRC* catalyzes the dissociation of the trimeric G protein (Gαβγ) into an activated α-subunit (Gα*) and the β- and γ-subunits (Gβγ). Gα* spontaneously deactivates (Gα) and reassembles with Gβγ, or it reversibly interacts with the adenylate cyclase (AC) to form an enzymatically active complex (Gα*/AC*), which serves as the output. Following data from <i>Aplysia </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Yovell1" target="_blank">[41]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Onyike1" target="_blank">[43]</a>, Ca⁺⁺ in turn transiently increases the rate constants for both the formation and the dissociation of the Gα*/AC* complex (represented by the thickened arrows). The k_subscript denote the rate constants of the respective reactions. B. When this model is stimulated with a transmitter input alone the Gα*/AC* concentration rises to a peak of ∼0.42 molecules/µm² in ∼20 s after stimulus onset, and decays back to zero within the next ∼100 s (left). If a Ca⁺⁺ input immediately precedes the transmitter, the build-up of the Gα*/AC* concentration is transiently accelerated (middle). If on the other hand the Ca⁺⁺ input follows the transmitter, the decay of the Gα*/AC* concentration is transiently accelerated (right). For graphical reasons, normalized concentrations are calculated by dividing with the peak Gα*/AC* concentration given transmitter input alone. The transmitter concentration reaches a peak of ∼6.7·10⁴ molecules/µm² in ∼7 s and decays back to zero within ∼18 s; the Ca⁺⁺ concentration starts rising ∼4.5 s after the onset, reaches a peak value of 5.6·10⁻⁴ moles/L at ∼6 s and decays back to zero within ∼8.5 s after the onset. Also these inputs are plotted as normalized concentrations.</p

Components and initial concentrations for the first model.

<p>All values were chosen according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Rospars1" target="_blank">[46]</a> and were estimates from moth olfactory transduction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Rospars1" target="_blank">[46]</a> for further references).</p

Relative timing of the transmitter and Ca⁺⁺ affects the adenylate cyclase.

<p>We stimulate the model with transmitter and Ca⁺⁺ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g003" target="_blank">Fig. 3B</a> for the details). In the ‘control condition’ (left), Ca⁺⁺ precedes the transmitter by an onset-to-onset interval of 210 s. In ‘associative training’ (right), the two inputs follow each other with an inter-stimulus interval (ISI), which is varied across experiments. Negative ISIs indicate training with first Ca⁺⁺ and then the transmitter; positive ISIs mean the opposite sequence of inputs. For either condition, we take the area under the respective Gα*/AC* concentration curve as a measure of cAMP production. For each ISI, we calculate an ‘associative effect’, by subtracting the amount of cAMP produced during the respective associative training from that in the control condition. We then express the associative effect as percent of the area under the Gα*/AC* concentration curve in the control condition. These percent associative effects are plotted against the ISIs. For very large ISIs, we find no associative effect. If the Ca⁺⁺ is closely paired with the transmitter, we find negative associative effects; the strongest negative associative effect (−15.5%) is obtained when using ISI ∼−3 s. If on the other hand Ca⁺⁺ follows the offset of the transmitter during training, we find positive associative effects; the largest positive associative effect (6.3%) is obtained for ISI ∼26 s. Thus, depending on the relative timing of Ca⁺⁺ and transmitter during training, opposing associative effects come about, closely matching the behavioural situation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone-0032885-g001" target="_blank">Fig. 1</a>.</p

Event timing affects associative learning.

<p>Fruit flies are trained such that a control odour is presented alone, whereas a trained odour is paired with pulses of electric shock as reinforcement. Across groups, the inter-stimulus interval (ISI) between the onsets of the trained odour and shock is varied. Here, ISI is defined such that for negative ISI values, the trained odour precedes shock; positive ISI values mean that the trained odour follows shock. For each ISI, two fly subgroups are trained with switched roles for two odours (not shown). During the test, each subgroup is given the choice between the two odours; the difference between their preferences is taken as the learning index. Positive learning indices indicate conditioned approach to the trained odour, negative values reflect conditioned avoidance. Very long training ISIs support no significant conditioned behaviour. If the odour shortly precedes or overlaps with shock during training (ISI = −45 s, −15 s or 0 s), it is strongly avoided in the test (punishment learning). If the odour closely follows the shock-offset during training (ISI = 20 s or 40 s), flies approach it in the test (relief learning). *: <i>P</i><0.05/8 while comparing to zero in a sign test. Sample sizes are N = 8, 24, 34, 47, 24, 35, 12 and 12. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032885#pone.0032885-Yarali2" target="_blank">[15]</a>, with permission from Informa healthcare.</p