Figure 9. Relationship between individual simultaneously recorded eyelid molecular layer interneurons (MLIs) and eyelid Purkinje cells (PCs) during expression of conditioned eyelid responses.

(a and b) Peri-stimulus time histograms (10 ms bins) and raster plots sorted by eyelid conditioned response (CR) latency (red points) of the simultaneously recorded activity of eyelid PCs and MLIs. The dark gray bars and shaded areas indicate the duration of the tone stimulus (conditioned stimulus [CS]) and the black bars indicate the duration of the eyelid stimulation (unconditioned stimulus [US]). (c) Normalized instantaneous firing rate for the examples in a and b. For all three examples, the activity of the eyelid PC decreases before the simultaneously recorded activity of the eyelid PC-MLI increases, as well as prior to the time of onset of the conditioned response. The gray shaded region indicates the interstimulus interval (ISI) for each pair.

Figure 9—figure supplement 1. Relationship between activity of pairs of eyelid Purkinje cells (PCs) and putative PC-MLIs.

(a) Mean firing rate determined from all simultaneous recordings of PC and PC-MLI activity at three different interstimulus intervals (ISIs) (indicated by shaded rectangles). Mean PC-MLI activity (black lines) and PC activity (green lines) averaged across conditioned response (CR) trials illustrates how the inverse activity relationship of the pairs shifts to match the training interval; shading represents 95% confidence intervals. Gray lines represent activity measured during non-response trials. (b) Trial-by-trial variations in timing of activity of a PC and a putative PC-MLI simultaneously recorded during eyelid conditioning at different ISIs. The time of the decreases in PC activity and increases in putative PC-MLI activity were measured at 50% of their maximum responses and the difference between these values is plotted as the delay time. Positive numbers indicate trials where the eyelid PC reached its 50% response before the PC-MLI did, while negative numbers indicate that the PC-MLI reached its 50% response before the eyelid PC did. Red points indicate trials where a CR was observed, while blue points indicate trials where no CR was observed. The plots indicate that positive delays occurred whenever a CR was produced, while negative delays occurred whenever there was no CR. This indicates – on a trial-by-trial basis – that PC activity precedes putative PC-MLI activity during CRs. MLIs, molecular layer interneurons.

Figure 9—figure supplement 2. Quantification of temporal relationship for individual pairs of eyelid Purkinje cells (PCs) and PC-MLIs.

(a) Color-coded plots for each interstimulus interval (ISI) of the average normalized activity for all simultaneously recorded eyelid PC/PC-MLI pairs aligned to time of response onset (black dot). Green represents 100 ms before response onset and blue represents 100 ms after response onset in each case (inset). For each ISI, the trend was for PC activity to decrease before conditioned response onset and for the PC activity to continue to decrease while PC-MLI activity increased after response onset. (b) ΔPC and ΔMLI were calculated for each PC/PC-MLI pair whose activity was recorded simultaneously. For each pair, the change in PC activity (green) before response onset (PC preCR) was divided by the change over the entire duration (full PC); the same analysis was done for each PC-MLI response (black). (c) Comparison of preCR activity between eyelid PC/PC-MLI pairs. The ΔPC to ΔMLI ratio was plotted for each simultaneously recorded pair, while the diagonal line indicates equal changes before conditioned response onset. Points below the diagonal line indicate that the eyelid PC changed more than the simultaneously recorded activity of the putative PC-MLI, while deviations above the diagonal line indicate that the putative PC-MLI changed more than the eyelid PC before response onset. Each paired recording is color-coded according to the ISI (black = ISI 250, blue = ISI 500, red = ISI 700 and 750). MLIs, molecular layer interneurons.