|
Figure 4
Characterization under control conditions of the projections from the Hcrt neuron subpopulations in the pAH and aAH of 6 dpf transgenic Hcrt:EGFP zebrafish brains, along with an illustration of the Imaris software methodology used to quantify the branching, terminal points and sholl intersections of these projections. (a) Bar graphs (n = 5/group) show in control fish that the projections from pAH Hcrt neurons compared to those from aAH neurons have greater projection length and number of branch points, terminal points, and sholl intersections, with no Hcrt neurons or projections detected in the POA as indicated by flat bars in the graphs. (b) Photomicrograph (25×, dorsal view) obtained using confocal microscopy illustrates in a control Hcrt:EGFP zebrafish brain a representative image of the Hcrt neurons and projections (green) from the subpopulations, with the pAH, aAH and POA areas outlined by dashed lines and enlargements of a single representative Hcrt neuron along with its projections observed in the aAH (top) and pAH (bottom). (c) A digital representation created from the same confocal image using the “Filaments” function of Imaris software shows that the projections from Hcrt neurons located in the pAH (green) are primarily descending in the posterior direction, while the projections from Hcrt neurons located within the aAH (magenta) are primarily ascending in the anterior direction. The measures of projection length and number of branch points (white), terminal points (blue), and sholl intersections are derived from an analysis using Imaris “spots” of this digital reconstruction. Enlargements show with Imaris software the projections of a single representative Hcrt neuron located in the aAH (top) and pAH (bottom), with the white concentric circles starting at the projection point of origin of the Hcrt soma extending outwards to demonstrate the sholl intersection analysis, with the projection intersections through these circles used to measure the spatial distribution of these projections. Scale bars: low magnification 50 µm; high magnification 10 µm. Abbreviations: aAH: anterior part of the anterior hypothalamus, pAH: posterior part of the anterior hypothalamus, POA: preoptic area, Hcrt: hypocretin, dpf: days post fertilization.