Enhanced hippocampal noradrenaline and serotonin release in galanin-overexpressing mice after repeated forced swimming test

Abstract

Basal and forced swimming (FS) stress-induced release of noradrenaline (NA) and serotonin (5-HT) were determined by in vivo microdialysis in the ventral hippocampus of mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE/P) or under the dopamine beta-hydroxylase promoter (GalOE/D) (only NA). WT mice served as controls. Intraventricular infusion of galanin significantly reduced basal extracellular NA in WT mice and in GalOE/P mice (albeit less so). Microdialysis sampling during a 10-min FS showed that NA and 5-HT release were elevated to 213% and 156%, respectively, in the GalOE/P group, whereas in the WT group the increases were only 127% and 119%, respectively. The second (repeated) 10-min FS (RFS) caused a marked enhancement of NA and 5-HT release in the GalOE/P mice to 344% and 275%, respectively. However, the RFS caused only a 192% increase of extracellular NA levels in the GalOE/D mice. Pretreatment with the putative peptidergic galanin receptor antagonist M35 almost completely blocked the elevation of NA and 5-HT levels in the GalOE/P after RFS. These results suggest that the NA and 5-HT hippocampal afferents in GalOE/P mice are hypersensitive to both conditioned and unconditioned stressful stimuli, such as FS, and that this effect is mediated by galanin receptors. The present findings support a role of galanin in the regulation of release of NA and 5-HT, two neurotransmitters involved in mood control.

Fig. 1.

Infusion of porcine galanin (1 nmol per 0.5 μl per min i.c.v.) caused a gradual decrease of basal extracellular 5-HT and NA levels in the hippocampus of both WT and GalOE/P mice, measured over a period of 120 min. However, the 5-HT levels were reduced to a higher extent in the WT than in the GalOE/P mice, whereas the 5-HIAA concentrations were higher in the WT group. There was a significant difference between the AUC_{(0–120 min)} values for 5-HT and 5-HIAA in the GalOE/P mice (⋆⋆, P < 0.01) compared with the WT mice. In control experiments, the i.c.v. infusion of aCSF failed to alter basal NA and 5-HT concentrations (data not shown).

Fig. 2.

The effect of repeated 10-min swim stress on NA release in the hippocampus of WT and galanin-overexpressing mice. FS and, even more profoundly, RFS caused a marked increase in NA release in GalOE/P mice, which was significantly higher than the stress-induced NA release in the WT mice (*, P < 0.05; **, P < 0.01; ***, P < 0.002, n = 5) and the GalOE/D mice (, P < 0.05; , P < 0.01; , P < 0.002, n = 5). The galanin antagonist M35 given i.c.v. to GalOE/P mice significantly attenuated the elevation of NA levels induced by both FS and RFS stressors (, P < 0.05; , P < 0.001, n = 4–5). After FS, the AUC for NA in the GalOE/P mice was significantly higher than in the WT and GalOE/D mice (, P < 0.01, n = 5), whereas M35 completely abolished the stress effects induced by FS (, P < 0.001) and RFS (⋆⋆⋆, P < 0.001) in GalOE/P mice (Inset). M35 given to WT mice significantly reduced the AUC for NA (, P < 0.001; , P < 0.05; n = 5). The RFS caused a significant increase in AUC of NA in GalOE/P as compared to FS in the same group (⋆, P < 0.05) and compared to the AUC values after RFS in the WT and GalOE/D mice, respectively (⋆⋆⋆, P < 0.001).

Fig. 3.

The effect of repeated 10-min swim stress on 5-HT release in the hippocampus of WT and GalOE/P mice. FS and, even more profoundly, RFS caused significantly higher increase in 5-HT release in GalOE/P mice than in the WT mice (**, P < 0.01; ***, P < 0.002, n = 5), as also was confirmed by the AUC values (⋆⋆, P < 0.01) (Inset). The effect of RFS stress on 5-HT levels was almost completely blocked by the galanin antagonist M35 (+, P < 0.05, n = 3–5) at 130 min [★★★, P < 0.001 for the AUC_{(120–160 min)} values].

Fig. 4.

Immunofluorescence (A–D) and brightfield (E–H) micrographs of the hippocampal formation and cortex of WT/P (A and E), WT/D (B and F), GalOE/P (C and G), and GalOE/D (D and H) mice after incubation with galanin antiserum (A–D) or staining with cresyl violet (E–H). E–H show adjacent or semiadjacent sections to A–D, respectively. The various layers in the hippocampal formation and cortex are indicated in E.(A and B) A moderately dense galanin fiber network is seen in most layers in the WT mice (small arrowheads). Big arrowheads show strongly fluorescent fibers in stratum oriens. Note fluorescence in supragranular layer in A.(C) In the GalOE/P mouse, a strong galanin-LI can be seen in the granule cell layer (arrows) and in the mossy fibers (black arrowhead). Big arrowheads indicate strong fibers in stratum oriens, and small arrowheads indicate strong fluorescence in the superficial layer of the entorhinal cortex. Note that the moderately dense fiber network cannot be seen any longer. (D) Strong fluorescence is seen in the molecular layer and stratum lacunosum-moleculare (asterisk). Also note cell bodies in laminae II/III of the entorhinal cortex (arrows). Arrowheads show strongly fluorescent terminals in stratum oriens. CA1 and CA3, CA1 and CA3 pyramidal cell layers, respectively; GrDG, granule cell layer; LEnt, lateral entorhinal cortex; LMol, stratum lacunosum moleculare; Mol, molecular layer; Or, stratum oriens; PoDG, polymorph cell layer; PRh, perirhinal cortex; Rad, stratum radiatum; SLu, stratum lucidum. [Each bar indicates 200 μm (A for B–D; E for G; F for H).]

Fig. 5.

Immunofluorescence micrographs of the ventral hippocampal formation of WT/P (A), WT/D (B), GalOE/P (C), and GalOE/D (D) mice after incubation with galanin antiserum. Arrowheads indicate galanin-positive, presumably noradrenergic fibers forming a plexus of medium density (A, B, and D). Note that this immunoreactivity is almost absent in the GalOE/P mouse (C). (Bar = 100 μm for all micrographs.)