Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system

Abstract

Serotonin plays an important role in the regulation of anxiety states and physiological responses to aversive stimuli. Intracerebroventricular (i.c.v.) injection of the stress- and anxiety-related neuropeptide urocortin 2 (Ucn 2) increases c-Fos expression in serotonergic neurons in the dorsal (DRD) and caudal (DRC) parts of the dorsal raphe nucleus. These regions contain a subset of serotonergic neurons that projects via the dorsal raphe periventricular tract to periventricular structures, including the subfornical organ and ependymal layer, and to the ventricular system. To determine if Ucn 2 activates ventricle/periventricular-projecting serotonergic neurons in the midbrain raphe complex, we made i.c.v. injections of the retrograde tracer Fluoro-Gold into the lateral ventricle, followed 7 days later by i.c.v. injection of Ucn 2. The DRD at -8.18 mm and the DRC at -8.54 mm and -9.16 mm bregma were analyzed using a combined bright field and immunofluorescence technique. Approximately 40% of the ventricle/periventricular-projecting neurons in the subdivisions sampled were serotonergic. Urocortin 2 increased c-Fos expression in ventricle/periventricular-projecting serotonergic neurons in the DRC and in non-ventricle/periventricular-projecting serotonergic neurons in the DRD and DRC. Of the total population of ventricle/periventricular-projecting serotonergic neurons in the DRC at -8.54 and -9.16 mm bregma, 35% expressed c-Fos following Ucn 2 injections. These data are consistent with previous studies showing that i.c.v. injection of Ucn 2 activates subpopulations of serotonergic neurons restricted to the mid-rostrocaudal DRD and DRC and further demonstrate that these include both subsets of serotonergic neurons that do and do not project to the ventricle/periventricular system.

Figure 1

Photomicrographs illustrating Fluoro-Gold (FG) immunoreactivity following intracerebroventricular (i.c.v.) injection of FG into the lateral ventricle in Rat #135 (see also Figs. 5 and 6). A) Low power photomicrograph of a rat brain section from approximately −0.36 mm bregma showing FG-like immunoreactivity in the lateral ventricle (LV) and the rostral part of the third ventricle (3V) including the vascular organ of the lamina terminalis (VOLT). Black box in A indicates region shown at higher magnification in D. B) Low power photomicrograph of a rat brain section from approximately −1.00 mm bregma, close to the injection site, including the subfornical organ (SFO). Black box in B indicates the region shown at higher magnification in E. C) Low power photomicrograph showing FG-like immunoreactivity in the 3V from approximately −3.30 mm bregma. Boxes in C indicate regions shown at higher magnification in F and G. Arrowheads in F and G show FG-like immunoreactivity in fibers (presumed tanycytes) close to the 3V. Abbreviations; 3V, third ventricle; bv, blood vessel, cc, corpus callosum; LV, lateral ventricle; SFO, subfornical organ; vhc, ventral hippocampal commissure; VOLT, vascular organ of the lamina terminalis. Scale bar, 500 μm (A,B), 250 μm (C), 100 μm (D,E), 50 μm (F,G).

Figure 2

Intracerebroventricular (i.c.v.) injection of mouse urocortin 2 (Ucn 2) increased c-Fos expression in specific subpopulations of tryptophan hydroxylase-immunoreactive (TPH-ir) neurons within the midbrain raphe complex. Closed bars represent the numbers of c-Fos-immunoreactive (c-Fos-ir)/TPH-ir neurons within each subdivision. Open bars represent the total numbers of TPH-ir neurons within each subdivision. The subdivisions and their rostrocaudal levels (mm bregma) are illustrated in line drawings from a standard stereotaxic atlas of the rat brain (Paxinos and Watson, 1998) above each column of graphs. Grey shaded areas in the line drawings highlight regions that showed increased c-Fos expression in serotonergic neurons following Ucn 2 injection. *p < 0.05, **p < 0.01 versus saline-treated controls, Fisher’s Protected LSD tests, (n = 7 for both groups). Abbreviations: DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; MnR, median raphe nucleus; Ucn 2, mouse urocortin 2; VLPAG, ventrolateral part of the periaqueductal gray.

Figure 3

Photomicrographs illustrating c-Fos expression in serotonergic neurons in the caudal part of the dorsal raphe nucleus (DRC; −8.54 mm bregma) following i.c.v. injection of Ucn 2. A) Representative photomicrograph of a section from an i.c.v. saline-injected control rat. B) Representative photomicrograph of a section from an i.c.v. Ucn 2-injected rat. Black boxes indicate regions shown at higher magnification in insets in the lower right corner of each image. Arrows indicate examples of c-Fos-ir cells (blue/black nuclear staining); white arrowheads indicate TPH-ir/c-Fos-immunonegative (serotonergic) neurons (brown/orange cytoplasmic staining); filled arrowheads indicate TPH-ir/c-Fos-ir (double-immunostained) neurons. Abbreviation: Aq, cerebral aqueduct; DRC, dorsal raphe nucleus, caudal part; DRI, dorsal raphe nucleus, interfascicular part; mlf, medial longitudinal fasciculus. Scale bar, 100 μm (A,B), 25 μm (insets).

Figure 4

Graphs illustrating the effects of i.c.v. Ucn 2 on c-Fos expression in ventricle/periventricular-projecting and non-ventricle/periventricular-projecting serotonergic neurons in the dorsal part of the dorsal raphe nucleus (DRD; −8.18 mm bregma; left column) and the caudal part of the dorsal raphe nucleus (DRC; −8.54 mm bregma; middle column; −9.16 mm bregma; right column). A) i.c.v. injection of Ucn 2 increased c-Fos expression in FG+ (ventricle/periventricular-projecting) neurons that were immunopositive for TPH (serotonergic neurons) in the DRC (−8.54 mm and −9.16 mm bregma) (closed bars). Open bars show the total numbers of FG+/TPH-ir neurons, closed bars show the numbers c-Fos-ir/FG+/TPH-ir (triple-labeled) neurons. B) i.c.v. injection of Ucn 2 increased c-Fos expression in FG− (non-ventricle/periventricular-projecting) serotonergic neurons in the DRD and DRC (closed bars). Open bars represent the total numbers of TPH-ir neurons. C) Graphs illustrating the proportion of FG+ (ventricle/periventricular-projecting) neurons that were serotonergic. Open bars show the total numbers of FG+ neurons, closed bars show the numbers of FG+/TPH-ir neurons. Note differences in y-axis scales. *p < 0.05, **p < 0.01 versus saline-injected controls, Fisher’s Protected LSD tests (n = 7 for both groups).

Figure 5

Brightfield, epifluorescence and confocal fluorescence photomicrographs illustrating ventricle/periventricular-projecting serotonergic neurons activated by i.c.v. injections of Ucn 2 (Rat#135, see also Figs. 1 and 6). A) Brightfield image of c-Fos-ir nuclei (brown/orange nuclear immunostaining) in the DRC (−8.54 mm bregma). Black box in A indicates the region shown at higher magnification with epifluorescence photomicrograph in B. White box in A indicates the region shown at higher magnification with a brightfield photomicrograph in C and in confocal fluorescence photomicrographs in D, E and F. The cerebral aqueduct is located at the top of the photomicrograph. B) Epifluorescence photomicrograph showing TPH-ir neurons (red). White box in B indicates the region shown at higher magnification in C-F. C) Brightfield photomicrograph showing two cells with c-Fos-ir nuclei (white arrowheads). D) Confocal autofluorescence image of FG+ neurons (DAPI). E) Confocal immunofluorescence image of TPH-ir neurons (Cy5). F) Combined confocal image of DAPI and Cy5 showing TPH-ir/FG+ neurons. Note that nuclear c-Fos immunoreactivity occludes the DAPI/Cy5 fluorescence. White arrowheads show examples of c-Fos-ir/FG+/TPH-ir (triple-labeled) neurons. Abbreviations: BF, brightfield; DRC, dorsal raphe nucleus, caudal part. Scale bar, 72 μm (A), 37 μm (B), 25 μm (C–F).

Figure 6

Schematic illustration showing the distribution of c-Fos expression in ventricle/periventricular-projecting serotonergic and non-serotonergic neurons in the dorsal raphe nucleus (Rat#135, see also figures 1 and 5). A) DRD (−8.18 mm bregma). B) DRC (−8.54 mm bregma). C) DRC (−9.16 mm bregma). Black circles represent c-Fos-immunonegative/FG+/TPH-ir neurons, white circles represent c-Fos-immunonegative/FG+/TPH-immunonegative neurons, grey square in B shows a c-Fos-ir/FG+/TPH-immunonegative neuron and black triangles show c-Fos-ir/FG+/TPH-ir (triple-labeled) neurons. Abbreviations; Aq, cerebral aqueduct; DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DTgP, dorsal tegmental nucleus, pericentral part. Scale bar, 50 μm.