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Fig. 1 | BMC Biology

Fig. 1

From: Characterization of a thalamic nucleus mediating habenula responses to changes in ambient illumination

Fig. 1

Overview of the habenula response to light ON and OFF. a, b Dorsal view of the fore and midbrain of 5-day-old elavl3:GCaMP6f fish, imaged with wide-field fluorescence microscopy at 200 Hz. The time since start of illumination is shown at the top right. The wedge indicates the ratio of fluorescence relative to the first frame. There is an increase in GCaMP6f fluorescence in the left habenula (arrowhead in b). c Maximum ΔF/F0 value in the left and right habenula after onset of light in five different fish. Each circle is one fish and the line joins data points from the same fish. df Two-photon imaging of the habenula in a 7 dpf GAL4s1011t, UAS:GCaMP6s fish, at 13 Hz. d Average of the time-lapse sequence, showing anatomy. The dorsal left neuropil is indicated by the yellow arrowhead. e Spatial distribution of responses to pulses of light. Pixels are color-coded according to the temporal pattern of response, as indicated in panel f. f Centers of clusters obtained from running K-means on the time series of pixels in panel d. Cluster centers are colored in shades of blue for responses to light ON and magenta and orange for responses to light OFF. The presence of light is indicated by the blue bars. g, h Neuropil response summarized from imaging 10 fish exposed to 7 pulses of blue light. g Pixels in the left neuropil from all fish could be classified into three main classes. They are pseudo-colored and overlaid on an average image of a 6 dpf fish. The largest response was a transient response to light ON (blue). A sustained response to light ON (cyan) and OFF (magenta) were also seen. Responses were reproducible in all 10 fish. h Average traces obtained from neuropil pixels, shown here for two pulses of light. i Percentage of cells active to light ON and OFF in the habenula is correlated with the percentage of active pixels in neuropil. The transient and sustained neuropil responses were combined into ON. Percentage of active cells or pixels were calculated by dividing the number of cells/pixels active to the stimulus by the total number of segmented cells or neuropil pixels. Each circle per category is one fish. The bold lines show best fit (linear regression), r is the correlation coefficient. jk Cumulative probability of peak ∆F/F0 response in cells and neuropil pixels responding to light ON (j) and light OFF (k). The response in the neuropil precedes the response in cells. P value and test statistic (D) were obtained by a Kolmogorov–Smirnov test between categories in the first 5 seconds. In panel j, the diamond ♦  (left) is the result of comparison between neuropil transient On and cell On, while plus + (right) between neuropil sustained On and cell On. rHb right habenula, lHb left habenula, Pa pallium, OT optic tectum, Pi Pineal, a anterior, p posterior. Scale bar = 25 μm

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