A role for galanin in antidepressant actions with a focus on the dorsal raphe nucleus

Abstract

Selective serotonin reuptake inhibitors, such as fluoxetine (FLX), are the most commonly used drugs in the treatment of major depression. However, there is a limited understanding of their molecular mechanism of action. Although the acute effect of selective serotonin reuptake inhibitors in elevating synaptic serotonin concentrations is well known, the clinical amelioration of depressive symptoms requires 14-21 days of treatment, suggesting that numerous other rearrangements of function in the CNS must take place. In the present study, we demonstrated that 14 days of FLX treatment up-regulated galanin mRNA levels by 100% and GalR2-binding sites by 50%, in the rat dorsal raphe nucleus, where galanin coexists with serotonin. Furthermore, a galanin receptor antagonist, M40, attenuated the antidepressant-like effect of FLX in the forced swim test, a rodent preclinical screen commonly used to evaluate antidepressant-like efficacy. Direct activation of galanin receptors by a galanin receptor agonist, galnon, was found to produce an antidepressant-like effect in the same task. Two other antidepressant treatments also affected the galaninergic system in the monoaminergic nuclei: Electroconvulsive shock elevated galanin mRNA levels in dorsal raphe nucleus, whereas sleep deprivation increased galanin mRNA levels in the locus coeruleus, further underlining the connection between activation of the galaninergic system and antidepressant action of various clinically proven treatments.

Fig. 1.

Galanin-like immunoreactivity in the rat DRN and LC. (A) Immunofluorescent double labeling of tryptophan hydroxylase (TPH, in green) and galanin (GAL, in red) in DRN. (B) Immunofluorescent double labeling of dopamine β-hydroxylase (DBH, in green) and galanin (in red) in the LC. (Scale bar, 50 μm).

Fig. 2.

Effects of antidepressant treatments on galanin mRNA expression. Adult male rats were subjected to one of the three antidepressant treatments, i.e., 24-h sleep deprivation, electroconvulsive shock (four shocks daily for 2 days), or FLX (10 mg/kg i.p. for 14 days). Different brain regions were dissected and tissues from 6-12 rats were pooled. Total RNAs were extracted and reverse transcribed with oligo(dT) primers. Real-time PCRs were repeated three times on pooled samples (A and B). (A) Galanin mRNA in different regions of naive rat brain was quantified and expressed as arbitrary unit. (B) Antidepressant treatments increased galanin mRNA expression in various brain regions. (C) FLX treatment increased galanin mRNA in the DRN and LC. Additional experiments were conducted on three independent pools of four rats/region/treatment to verify the effects of chronic FLX administration on galanin mRNA levels in the DRN and LC. ^*, P < 0.05, Ctrl vs. FLX, Student's t test. PFC, prefrontal cortex; Amy, amygdala; Hip, hippocampus; PVN, paraventricular nucleus of hypothalamus; SD, sleep deprivation; ECS, electroconvulsive shock.

Fig. 3.

Chronic FLX treatments up-regulate GalR2 protein levels in DRN. After electroconvulsive shock (four shocks per day, 2 days) or chronic (14 days) FLX (10 mg/kg, i.p.) treatment, the levels of GalRs in DRN and LC were determined with saturating [¹²⁵I]galanin binding (1 nM). Each treatment group included 16 rats, and samples from 3 animals were combined. Total galanin-binding sites (the sum of GalR1 and GalR2) and GalR2 sites were estimated by using 5 μM galanin (1-29) and 5 μM galanin (2-11) as competitors, respectively. Because GalR3 is not abundant in these regions, the difference between the total GalRs and GalR2 was defined as GalR1. The results are expressed as femtomoles per milligram protein ± SEM. Chronic FLX treatment increased GalR2 sites in DRN but not in LC (B). None of the antidepressant treatments has effects on GalR1 sites (A). ^*, P < 0.05, Bonferroni t test for multiple comparisons with single control group.

Fig. 4.

Galanin receptor antagonist, M40, attenuated antidepressant-like effect of FLX in the forced swim test. (A) Displacement of [¹²⁵I]galanin binding by M40 from membranes prepared from GalR1- and GalR2-expressing cells. M40 has similar affinity for GalR1 and GalR2 with K_i values of 1.8 nM and 5.1 nM, respectively. (B) Results from rats pretreated for 14 days with FLX (10 mg/kg) or saline and given single i.c.v. infusion of the galanin receptor antagonist M40 or vehicle (ACSF) 15 min before testing in the forced swim test. Activity was measured during a 10 min test. Data represent group means (± SEM) of percentage of time spent active in forced swim test. ^*, Significance between saline/ACSF vs. FLX/ACSF. Rats pretreated for 14 days with FLX exhibited an ≈46% increase in time spent active in the forced swim test compared to saline-pretreated rats; this effect was significant [P < 0.05, Fisher's least significant difference (LSD) test]. This effect of FLX pretreatment was completely reversed by i.c.v. administration of M40. ^**, Significance of FLX/ACSF vs. FLX/M40, P < 0.01, Fisher's LSD.

Fig. 5.

Effects of treatment of rats with different doses of galnon i.p. in the forced swim test and open field test. (A) Activity was measured in the forced swim test during a 10-min test at 45 min after injection. Higher doses of galnon produced a significant increase in activity in the test, compared to vehicle treated animals. As a positive control, one group was treated with the tricyclic antidepressant desipramine (15 mg/kg, i.p.), which also displayed significantly increased activity. (B) Identical doses of drugs were administered in the open field test. Higher doses of galnon and desipramine significantly reduced activity in this task. (^*, P < 0.05 vs. control, Fisher's least significant difference test).