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. 2007 Jul 4;27(27):7344–7360. doi: 10.1523/JNEUROSCI.0873-07.2007

Figure 6.

Figure 6.

A, B, Example of a hypothalamic slice used in this study for evaluating NE actions on the PVH in vitro. A, A low-power view of a hemislice used in an early control experiment, with the area outlined by a rectangle shown in higher magnification in B. Note the locations of the fornix (f), third ventricle (3), as well as the faint presence of clustered cells that mark the location of the PVH, the outer boundary of which is estimated by the dashed line. In B, the arrowheads mark the course of a blood vessel entering the PVH, which originated from the upper margin of the optic tract (o) (A). Scale bars: A, 1 mm; B, 250 μm. These photographs were taken through the top cover glass of the “sandwich” holding the slice (see Materials and Methods). C–J, NE treatment triggers robust elevations in PVH phospho-ERK in coronal hypothalamic slices maintained in vitro. Single-plane, 10× (C, D) and 20× (E–J) confocal images of the PVH on one side of the midline are shown. Slices received either no treatment (Control, aCSF bath only; C–I) or 200 μm NE treatment (D–J) and were then fixed in 4% paraformaldehyde 10 min later. C and D show overlays of CRH immunoreactivity (green) and phospho-ERK1/2 immunoreactivity (red). Note that control cases show only blood vessel labeling in the red channel (C, arrow), whereas NE-treated cases show phospho-ERK1/2 in the PVH but not surrounding area (D). CRH immunoreactivity is sometimes apparent in neurites extending from PVH cell bodies (E, arrowheads), and phospho-ERK1/2 is seen in cell bodies of NE-treated slices (H), but not control slices (G). Colocalization of CRH and phospho-ERK1/2 appears in NE-treated slices (J) as colored signal ranging from yellow to orange, which is not evident in control slices (I). For some readers the colocalization signal is more apparent if red is pseudocolored to magenta; this can be seen in supplemental Figure S3 (available at www.jneurosci.org as supplemental material). The boxes in I and J outline regions analyzed through the z-axis, photomicrographs for which are shown in Figure 7, A and B, respectively. Scale bars: (in C) C, D, 100 μm; (in E) E–J, 50 μm. In D, f is fornix.