Evidence for intact 5-HT1A receptor-mediated feedback inhibition following sustained antidepressant treatment in a rat model of depression

Abstract

Serotonin (5-HT) neurons are strongly implicated in mood disorders such as depression and are importantly regulated by feedback inhibition mediated by 5-HT_1A receptors. These receptors may play a role, albeit a poorly understood one, in the generation of mood disorders, treatment response to antidepressants and delayed therapeutic efficacy. Here we sought to gain insight into the role of 5-HT_1A receptor-mediated feedback inhibition in these processes by studying Fos protein expression within serotonin neurons in a rat model of stress-related mood disorder, early life maternal separation (MS), combined with two-week treatment with the antidepressant fluoxetine (FLX) in adulthood. We gauged 5-HT_1A receptor-mediated feedback inhibition by the ability of the antagonist, WAY-100635 (WAY), to disinhibit Fos expression in 5-HT neurons. We found that two-week FLX treatment dramatically inhibited Fos expression in serotonin neurons and that this effect was reversed by blocking 5-HT_1A receptors with WAY. Together these observations reveal that after prolonged exposure to SSRIs, endogenous 5-HT_1A receptors continue to exert feedback inhibition of serotonin neurons. Furthermore we found unique effects of pharmacological treatments after MS in that the WAY effect was greatest in MS rats treated with FLX, a phenomenon selective to the rostral 2/3 of the dorsal raphe nucleus (B7). These results indicate that the balance between activation and feedback inhibition of serotonin neurons in B7 is altered and uniquely sensitive to FLX after early-life stress.

Keywords: Fluoxetine; Fluoxetine hydrochloride (PubChem CID: 62857); Fos; Raphe; Serotonin; Stress; Swim; WAY-100635 (PubChem CID: 5684).

Figure 1.

Experimental design and raphe subdivisions analyzed. A) Schematic depicting experimental design. B) Group sizes and labels for all experimental conditions. C) Rostral dorsal raphe, dorsal (RD) and ventral (RV), sampled between Bregma −6.92mm to −7.64mm. Scale bar = 100 μm. D) Mid dorsal raphe, dorsal (MD), ventral (MV), and lateral wings (LW), sampled between Bregma atlas coordinates −7.73mm to −8.45mm. Scale bar = 200 μm. E) Caudal dorsal raphe, dorsal (CD) and ventral (CV), sampled between Bregma atlas coordinates −8.54mm to 9.26mm. Scale bar = 100 μm. F) Median raphe (MR) was also sampled at the mid dorsal raphe level. Data obtained from RD, RV, MD, MV, and LW (B7) and CD, CV, and MR (B6 + MR) were pooled for analysis. Single images were stitched using FIJI (Preibisch et al (2009) Bioinformatics 25: 1463–5). Scale bar = 100 μm.

Figure 2.

Fluoxetine (FLX) treatment reduces Fos activation within serotonin neurons. A) Two weeks of FLX treatment dramatically decreased Fos expression in serotonin neurons of the rostral dorsal raphe (B7) and in the caudal dorsal and median raphe (B6 + MR). B) Sub-regional analysis revealed that FLX robustly decreased FOS across all sub-regions. All data in A) and B) are expressed as group means ± 1 SEM, and an asterisk indicates statistical significance (p<0.05) compared to vehicle-treated rats. C) Representative images depicting immunofluorescent labeling for tryptophan hydroxylase only (TPH; green), Fos only (magenta), or both proteins overlaid (merged) from the mid-ventral (MV) subregion of the dorsal raphe of a vehicle-treated (VEH) and a fluoxetine-treated (FLX) rat. White arrows indicate double-labeled cells. Scale bar = 50 μm.

Figure 3.

Acutely antagonizing 5-HT_1A receptors reverses the effect of fluoxetine (FLX) treatment, demonstrating retained 5-HT_1A receptor-mediated feedback inhibition. A) Acute treatment with the 5-HT_1A receptor antagonist WAY-100635 (WAY) completely reversed the suppressing effect of repeated FLX treatment on Fos expression in serotonin neurons within B7 and B6 + MR raphe nuclei. Both with or without repeated treatment with FLX, acute WAY treatment significantly increased serotonin neuron Fos expression compared to vehicle (VEH) treatment in the rostral dorsal raphe (B7). In B6 + MR, WAY and VEH treated groups had equivalent levels of Fos, whereas WAY reversed the effect of FLX to VEH levels. * = p<0.05 compared to CON VEH group. B) Examining these effects with more regional granularity, WAY treatment alone significantly increased Fos in serotonin neurons in the rostral (B7) raphe subregions RD, RV, and LW. * = p<0.05 compared to CON VEH group. C) In rats treated with FLX, WAY dramatically increased Fos in serotonin neurons in all sub-regions examined, demonstrating WAY-mediated disinhibition of raphe 5-HT neurons. * = p<0.05 compared to CON-FLX group. D) WAY treatment produced similar FOS expression in serotonin neurons regardless of FLX treatment in almost all raphe sub-regions. In the caudal ventral sub-region (CV), WAY treatment alone induced significantly more Fos in serotonin neurons than WAY treatment in combination with FLX treatment, * = p<0.05 compared to CON FLX/WAY group, although neither treatment group differed from CON-VEH in this region

Figure 4.

Maternal separation (MS) influences 5-HT_1A receptor-mediated feedback inhibition in the rostral dorsal raphe (B7), but not the caudal dorsal raphe and median raphe (B6 + MR). A) Acutely blocking 5-HT_1A receptors with WAY after fluoxetine (FLX) treatment had a larger effect in the B7 of MS rats than in control-reared (CON) rats. * = p<0.05 compared to VEH group. # = p<0.05 compared to WAY group. B) In the B6 + MR raphe sub-regions, the effects of FLX and WAY were similar in CON and MS rats. * = p<0.05 compared to VEH group. C) In control rats, WAY treatment led to similar FOS expression in all raphe regions analyzed (except for the caudal ventral; CV) regardless of FLX treatment (same data as shown in Fig. 3D). * = p<0.05 compared to WAY group. D) In comparison, in MS rats combined FLX and WAY treatment (FLX/WAY) led to significantly higher FOS in serotonin neurons in the rostral dorsal raphe (B7) compared to WAY treatment alone, although FOS expression was similar in these two groups in the caudal dorsal and median raphe (B6 +MR). * = p<0.05 compared to WAY group.