Figure 1.
β – galactosidase staining is found throughout the brainstem, but is especially dense within the nTS and hypoglossal nucleus.
Left: Three rostrocaudal nTS levels designated as Rostral, Intermediate, and Caudal. Coordinates from Bregma are listed for each nTS level. Numbers in parenthesis represent coordinates relative to the rostral-most (i.e. ‘r1’) portion of the nTS as defined previously [17]. AP: area postrema, CC: central canal, ECu: external cuneate nucleus, icp: inferior peduncle, nTS: nucleus of the solitary tract, py: pyramidal tract, sp5: spinal trigeminal tract, Sp5n: spinal trigeminal nucleus, T: solitary tract, 4V: 4th ventricle, 10n: dorsal motor nucleus of the vagus, 12n: hypoglossal nucleus. Images modified from Paxinos The Mouse Brain in Stereotaxic Coordinates, 2nd Edition. Right: Photomicrographs of fluorescent β – galactosidase staining in the brainstem of a FTL mouse that received no stimulation (Unstim) Images converted to greyscale colors for clarity.
Figure 2.
β – galactosidase staining is similar across experimental conditions; whereas c-Fos protein label varies significantly.
Left: β-gal staining (green) is not different across all experimental groups. Middle: Significant c-Fos staining (magenta) is present only in the MSG group. However, punctate c-Fos staining (red arrow) is found in all other groups. Moreover, c-Fos protein staining is found in the cellular processes of some c-Fos labeled cells in the MSG group (inset). Right: A significant proportion of doubled labeled c-Fos/β-gal cells (white label and left most yellow arrow) is found in the MSG group. Interestingly, we observed some c-Fos positive cells that were not co-labeled with β-gal (middle yellow arrow) as well as some β-gal positive cells that did not co-localize with the c-Fos protein (right yellow arrow). Sections shown are of the ‘Rostral’ representative level.
Figure 3.
Bar graphs summarizing all quantative measurements across experimental conditions.
A: The number of β-gal positive cells (green) was not different within each nTS level (Rostral, Intermediate, Caudal), nor between stimulation groups (Unstim, No Food/Water, MSG, MSG 5 h Post, p = 0.25). Interestingly, the number of β-gal positive cells, irrespective of group, was significantly different between nTS levels (Rostral>Intermediate>Caudal; all p's>0.05, data not shown). B: In contrast to the number of β-gal positive cells, the number of c-Fos positive cells (Fos-LI; magenta) was significantly different between groups, with Fos-LI in the MSG group greater than all other groups. Also, within the nTS of the MSG group, Fos-LI was significantly different between nTS levels (Intermediate (Intermed.)>Rostral>Caudal). C: The number of c-Fos/β-gal double labeled cells matched the Fos-LI results seen in C, with the number of double labeled cells significantly greater in the MSG group as compared to all other groups. Also, the number of double labeled cells was significantly greater in the Rostral level of the nTS in the MSG group compared to both the Intermed. and Caudal groups. D: The number of β-gal (green) labeled pixels that exceeded threshold (which includes both cell bodies and cellular processes) was not statistically different between groups (p = 0.36). However, the number of c-Fos labeled pixels (magenta) was significantly greater in the MSG group as compared to all other groups. In all figures: n.s. = not statistically significantly different; * = a significant difference between the MSG group vs. all other groups; # = a significant different between nTS levels within the MSG group. All p's<0.05.