The thalamus drives light-evoked activity in the habenula of larval zebrafish

larval zebrafish 2 Ruey-Kuang Cheng, Seetha Krishnan, Qian Lin, David G. C. Hildebrand , Isaac H. 3 Bianco, Caroline Kibat and *Suresh Jesuthasan 5, 6, 7 4 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 5 636921. 6 NUS Graduate School for Integrative Sciences and Engineering, 28 Medical Drive, 7 National University of Singapore, Singapore 117456. 8 Graduate Program in Neuroscience, Division of Medical Sciences, Graduate School of 9 Arts and Sciences, Harvard University, Cambridge, Massachusetts 02138 United States 10 of America. 11 Department of Molecular and Cell Biology, Harvard University, Cambridge, 12 Massachusetts 02138, United States of America. 13 Neural Circuitry and Behavior Laboratory, Institute of Molecular and Cell Biology, 14 Singapore 138673. 15 Neuroscience and Behavioral Disorders Program, Duke-NUS Graduate Medical School, 16 8 College Road, Singapore 169857. 17 Department of Physiology, National University of Singapore, Singapore 117597. 18

The habenula is an evolutionarily conserved structure (Stephenson-Jones et al., at a rate of 1 Hz. Habenula activity was monitored as the larva was exposed to 20-8 4 second pulses of blue light. We used relatively long pulses, rather than brief flashes, to 8 5 allow responses to transition as well as steady state to be identified. Pixel-wise analysis and to light and darkness ( Figure 1D, E). The spatio-temporal pattern of activity was 8 9 reproducible across several cycles, as shown by the trajectory of the system through 9 0 state space ( Figure 1F).

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To assess if these responses were reproducible across multiple fish, we imaged The s1011tGAL4 line drives GCaMP3 expression throughout the habenula, and We next searched for afferents that could provide such properties in larval 1 3 9 zebrafish. We focused initially on inputs that could account for the asymmetry in the  T  h  e  h  a  b  e  n  u  l  a  m  a  y  r  e  c  e  i  v  e  g  l  u  t  a  m  a  t  e  r  g  i  c  a  n  d  G  A  B  A  e  r  g  i  c  i  n  p  u  t  f  r  o  m  t  h  e  t  h  a  l  a  m  which may mediate light-evoked excitation and inhibition of habenula neurons. If the thalamus provides afferents mediating illumination-dependent activity in the the thalamus has a response to both increase and decrease of illumination. here should depend on the eyes. Indeed, the robust responses to light were lost in fish   As shown in Figure 4G-I, light evokes strong activity in the dorsal neuropil of the to the neuropil of the putative nucleus rostrolateralis, which was identified by first 2 2 7 imaging the response to light pulses ( Figure 9A, B). Lesioning led visible damage in the 2 2 8 neuropil ( Figure 9D), and to a reduction of evoked activity in the thalamus and habenula hypothesis that the putative nucleus rostrolateralis of the thalamus has a role in light-2 3 5 evoked activity in the habenula of larval zebrafish. Light is a potent regulator of brain function. It can affect mood (Vandewalle et al., including intrinsically-sensitive retinal ganglion cells whose targets include the thalamus of these effects of light may be mediated by the thalamic projection to the habenula.

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A projection from the thalamus to the habenula may be evolutionarily conserved in vertebrates. In humans and rabbits, a thalamo-habenula projection was proposed many 2 9 7 years ago based on degeneration experiments (Marburg, 1944;Cragg, 1961). Using geniculate nucleus and other thalamic nuclei to the habenula. It will be interesting to   wildtype. Tg(elavl3:GCaMP6f)a12200 was generated by PCR amplification of the GCaMP6f ng/μL. A single founder was selected based on high and spatially broad expression.

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Outcrossing this founder generated 50% GCaMP6f-positive embryos, which were 3 2 9 selected to establish the line. Zebrafish larvae (aged 5 -10 dpf) were anaesthetized in mivacurium and a fixed stage upright microscope, using a 25x water immersion objective (NA = 1.1). The femtosecond laser (Coherent Vision II) was tuned to 920 nm for GCaMP imaging. Stacks Light stimuli were generated by 5 mm blue LEDs (458 nm peak emission). They   directly analysed as pixels (see Below).

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Correlation to light evoked activity: Temporal traces from pixels (Thalamus or optimal k of 6-10; 3-4 clusters that didn't correspond to evoked activity were not included were also plotted here. To verify the results of k-means, the presence of light response  The scripts used for analysis are provided at http://dx.doi.org/10.5061/dryad.q0171. then maximally projected to a single image, which was then subjected to a minimum filter 4 1 0 and unsharp mask to sharpen the boundary of cells. ROIs were identified using the "find 4 1 1 maxima…" command, as a way to localize regional darkest point as the center of each the algorithm (<10% of total ROIs). In the last step, "Set measurements…" and which z drifting occurred were excluded, as in this case ROIs could not be defined. detrended, smoothed and normalized to z-scores using baseline as the time before the 4 2 7 first blue light. Traces that did not reach a Z-score of 2 during the period of irradiance  DiD (Life Technologies) was dissolved in 50 µl ethanol to make a saturated 4 4 7 solution. This was heated to 55˚C for 5 minutes prior to injection into the fish that had were stored at 4˚C overnight to allow tracing, and then imaged with a 40x water immersion objective on a Zeiss LSM 710 confocal microscope. Larvae were fixed in 4% para-formaldehyde/PBS overnight at 4˚C. They were then 4 5 7 rinsed in PBS. The brains were dissected out, and permeabalized using 1% BSA goat anti-rabbit; 1:1000). After washing, these were mounted in 1.2% agarose/PBS.

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Imaging was carried out using a Zeiss LSM 510 laser scanning confocal microscope, with a 40x water immersion objective. MS222. This procedure was carried out in Ringers saline. Fish were then mounted in 4 7 0 1.2% agarose in Ringers saline, and imaged using two-photon microscopy as described provided, with a pulse duration of 25 milliseconds and a frequency between 1 and 8 Hz. Each fish was exposed to at least 3 pulse trains. For Figure 6B-C, the average of the first 4 7 6 29 frames was used as a reference. The ratio of all frames relative to this reference was to the genotype. bleeding in the brain after lesioning, due to bursting of a blood vessel in the thalamis, 4 9 5 were discarded. The mouse connectivity atlas (http://connectivity.brain-map.org/) was searched in 4 9 8 ""Target Search" mode, using "TH" as the source structure and "EPI" as the target This work was funded by core funding from the Institute of Molecular and Cell 5 0 5 Biology and a grant from the Ministry of Education, Singapore. SK and QL were Neurosci 31:8708-8712.  Academy of Sciences 106:17968-17973. Chen T-W, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr  Thornton EW, Davies C (1991) A water-maze discrimination learning deficit in the rat    Cell 166:716-728. dimensional state space, using the first two principal components (PC1 and PC2). which change in irradiance drives the neural state. In panels E and F, the bold lines Segmentation of habenula neurons using a semi-automated algorithm (see Methods). corresponds to a single pixel. Panels below show the average of the heat maps above. The shaded region is standard error of mean. Blue boxes indicate when light was Habenula activity before (E) and after (G) lesion of the thalamic neuropil. Pixels are 8 3 7 colored according to the traces in (F). There is a reduction in the sustained response to AF7 (n = 2 fish) or AF9 (n = 3 fish), or before lesion (n = 5 fish). P-value was obtained 8 4 6 using non parametric Wilcoxon rank sum test. Z is the test statistic, and r is the effect 8 4 7 size. The statistical comparisons were made between before lesion and after lesion. a: sections. Scale bar = 25 µm. habenula. mHb: medial habenula. These images are from http://connectivity.brain-8 5 8 map.org/projection/experiment/siv/267538006?imageId=267538231&imageType=TWO_ 8 5 9 PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=14704&y=7847&z=3. interpeduncular nucleus (IPN), but not to the thalamus. to project to the neuropils of the habenula. Red label also appears in streaks in the 8 6 7 lateral habenula. Anterior is to the top. with the thalamus. S1011Et drives GAL4 expression in the habenula, medial pallium and 8 7 5 anterior-lateral pallium. This is a dorsal view, with anterior to the left. (magenta) are visible in the thalamus, below the habenula. Anterior is to the left. The stack goes from dorsal to ventral. There is a reduction in the sustained response to light, but some activity that is not stimulus-locked can be seen. (H) The habenula after lesion of AF9, with pixels colored according to the traces in panel (I).
(J-M) Heatmaps showing activity in segmented cells before (J) and after (K) AF9 lesion, and before (L) and after (M) thalamic neuropil lesion in one fish. Panels below show mean (black trace) and standard error of mean (shaded region). Light evoked activity is missing following this lesion. (N) Boxplot showing number of cells in one plane of the dorsal left habenula that are excited by blue light, following lesion of the thalamic neuropil (n = 12 fish), or AF7 (n = 2 fish) or AF9 (n = 3 fish), or before lesion (n = 5 fish). P-value was obtained using non parametric Wilcoxon rank sum test. Z is the test statistic, and r is the effect size. The statistical comparisons were made between before lesion and after lesion. a: anterior; p: posterior; Pa: pallium; rHb: right habenula. Images are all single optical sections. Scale bar = 25 µm.