Article Figures & Data
Figures
- Figure 1.
Classical conditioning in larval zebrafish. A, Conditioning protocol. In the first trial (pre) a single CS was presented to examine the animal's response to the CS alone. Ten paired CS+US trials were presented with a 6 min intertrial interval (ITI). Three test trials (CS alone) were presented after various delays. B, Sample analysis of conditioned and unconditioned responses (CR and UR, respectively). The tail angular velocity before (upper trace) and after (lower trace) learning was compared. The UR can be seen after US presentation (red arrows). The red line represents the CS. C, The effect of paired (CS+US), unpaired (CS+US unpaired), and CS alone trials on the CR. Each point represents the mean Vincrease value for all fish in a given trial ± SEM. Conditioning resulted in a significantly enhanced CR that grew in magnitude over the 10 trials and was rapidly extinguished by three test trials. Training with CS alone or unpaired CS+US did not significantly alter the CR. D, Memory decline over time. Fish were conditioned, and each group was tested twice (ITI = 4 min) either 5, 30, or 60 min after the last trial. Each bar represents mean Vincrease value for two trials ± SEM. *p < 0.05.
- Figure 2.
A, Change in fluorescence averaged across 25 light- and touch-evoked cerebellar neurons (green and red lines, respectively) as a function of time. The green arrow represents the onset of the visual stimulus (CS). The red arrow depicts the timing of touch delivery. B, Three-dimensional functional map of the left cerebellar hemisphere. The zero point on the z-axis is located at the dorsal surface of the cerebellum. Each z-section is separated by 10 μm. The intersection between x- and y-axes is the point where the midline between the hemispheres crosses the border to the optic tectum. Each circle depicts the position of an individual neuron. Green circles represent the light-driven neurons. Red circles represent touch-evoked neurons. Yellow circles represent neurons that responded to both visual and tactile stimuli. Orientation is as indicated: a, anterior; p, posterior; m, medial; l, lateral; d, dorsal, v, ventral. Histograms depict the density of light- and touch-evoked neurons (green and red bars, respectively) projected on x-, y-, and z-axes. Note that the density of touch-driven cells gradually increases in the dorsolateral direction, whereas light-evoked neurons are most densely packed at a depth of 40 μm.
- Figure 3.
Learning-dependent changes in visually driven activity. A, Neurons in the cerebellum (red) and optic tectum (blue) display relatively stable visual responses to a repetitively presented CS. B, Cerebellar, but not tectal, neurons, increase their responses to the CS during conditioning (CS+US). In A and B, each dot represents the response of an individual neuron (peak height) to the CS in trial 7 as a function of the response in trial 1. Color lines are linear trend lines. Bars on the right depict averaged responses in trials 1 and 7. ΔF/Fmin, percentage ± SEM; ***p < 0.001. C, Some neurons responded to the US only at the beginning of the training session but developed an additional peak of activity in response to CS after conditioning. An average response of 12 neurons from six fish (ΔF/Fmin, percentage ± SEM) in the first (blue) and seventh (green) trials is shown. Onsets of the CS and US are represented by the vertical red line and red arrow, respectively. *p < 0.05; **p < 0.01.
- Figure 4.
Requirement of the cerebellum, but not the telencephalon, for learning acquisition. A, Two-photon ablation of the telencephalon (left) and cerebellum (right). Top is a z-projection of confocal sections taken at depths of 0–40 μm. Bottom shows the left side of the same fish after ablation and the right side of a fixed brain stained with SYTO 14 (white rectangle shows the ablated region). Scale bar, 20 μm. B, Graph plots the CR (mean Vincrease ± SEM) as a function of trials. Bilateral ablation of the telencephalon performed immediately after the first (open red circles) or 10th (filled red circles) trial did not significantly impair acquisition or retention of memory. Bilateral lesions in the cerebellum after the first trial (open blue circles) blocked acquisition of the CR. Ablation after the 10th trial (filled blue circles) did not affect memory retention but impaired extinction during the three test trials.
