Fig. 6

Effect of eye removal on thalamus response to light ON and OFF. a–i Activity in the thalamus in response to pulses of blue light in control (n = 3) and enucleated (n = 4) fish. a, b Heatmaps showing activity in individual cells in control and enucleated fish classified as having response to light ON or OFF. c A comparison of the percentage of active cells in control and enucleated fish. The response to light ON is reduced in fish lacking eyes, while the response to light OFF is comparable to controls. d–e Histogram of mean response amplitude in cells in control and enucleated fish during (d) light ON and (e) OFF. Each trace is one fish. The amplitude of response to light ON is reduced in enucleated fish. Insets show cumulative histogram from all fish. f–i Pixels in the anterior thalamic neuropil of control (f) and enucleated (h) fish, that are active to light ON (cyan) or OFF (pink), were combined and overlaid. Panels f and h show a dorsal view of the thalamus. The average traces from the colored pixels in f and h are shown in g and i, respectively. Control fish have a response to light ON and OFF, whereas enucleated fish only have a response to light OFF. j Percentage of active neuropil pixels in control and enucleated fish. k–l Cumulative probability of mean response amplitude in pixels of control and enucleated animals to light ON (k) and OFF (l). Mean response during light OFF is not significantly different in enucleated and control fish. m Dorsal view of a 6-day-old fish, following injection of DiD into the dorsal left habenula neuropil and CM-DiI into the pineal organ. See Additional file 6: Movie 6 for the complete z-stack. n Lateral view of the right side of a 6-day-old fish, showing anterogradely labeled fibers from the pineal (red) and retrogradely labeled fibers from the habenula (cyan). The arrow indicates a pineal axon in the neuropil of the anterior thalamus. o, p Response in the pineal organ to pulses of blue light (n = 4 fish). Only OFF responses can be detected. (o) Pixels showing OFF responses are combined from all fish and overlaid. (p) Average trace from the colored pixels. The habenula is shown here for orientation only; habenula neuron responses have been masked. q–s Responses in the habenula to light OFF in control (q) and enucleated (r) fish (GAL4s1011t, UAS:GCaMP6s, n = 4 fish). Each row in the heatmaps represents an individual cell. s Percentage of cells showing an OFF response. Each circle is one fish and lines join data points from same fish before and after enucleation. Although reduced in number, there are still cells that display an OFF response. D-statistic and P values in panels d, e, k, and l were obtained using the Kolmogorov–Smirnov test on response amplitude distribution. Panels f, h, m, and o are single optical sections; n is a projection spanning 19.25 μm. rHb right habenula, lHb left habenula, Th thalamus, a anterior, p posterior, d dorsal, v ventral. Scale bar = 25 μm