Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus

Abstract

Recent studies suggest that stress-induced atrophy and loss of hippocampal neurons may contribute to the pathophysiology of depression. The aim of this study was to investigate the effect of antidepressants on hippocampal neurogenesis in the adult rat, using the thymidine analog bromodeoxyuridine (BrdU) as a marker for dividing cells. Our studies demonstrate that chronic antidepressant treatment significantly increases the number of BrdU-labeled cells in the dentate gyrus and hilus of the hippocampus. Administration of several different classes of antidepressant, but not non-antidepressant, agents was found to increase BrdU-labeled cell number, indicating that this is a common and selective action of antidepressants. In addition, upregulation of the number of BrdU-labeled cells is observed after chronic, but not acute, treatment, consistent with the time course for the therapeutic action of antidepressants. Additional studies demonstrated that antidepressant treatment increases the proliferation of hippocampal cells and that these new cells mature and become neurons, as determined by triple labeling for BrdU and neuronal- or glial-specific markers. These findings raise the possibility that increased cell proliferation and increased neuronal number may be a mechanism by which antidepressant treatment overcomes the stress-induced atrophy and loss of hippocampal neurons and may contribute to the therapeutic actions of antidepressant treatment.

Fig. 1.

Experimental paradigms. A,Proliferation and differentiation paradigms. Animals were administered antidepressants for 1–28 d. Four days after the last antidepressant treatment, animals were given BrdU and killed (S) 24 hr after BrdU administration. In one experiment designed to measure cell proliferation, animals were killed 2 hr after BrdU injection. To determine cell differentiation or phenotype, rats were killed 28 d after BrdU injection. B, Survival paradigm. To examine the influence of antidepressant treatment on the survival of BrdU-labeled cells, BrdU was given to drug-naïve animals before initiating antidepressant treatment (14 d). Rats were then killed 28 d after BrdU injection (14 d after the last antidepressant treatment).

Fig. 2.

The number of BrdU-positive cells in the adult hippocampus is increased after chronic antidepressant treatment. Rats received injections of BrdU 4 d after the last ECS (10 d) or drug (14–21 d) treatment and were killed 24 hr after the last BrdU injection. Shown are representative photomicrographs (10× magnification) from vehicle (A), tranylcypromine (B), ECS (C), or fluoxetine (D). The majority of the BrdU-labeled cells are located in the subgranular zone (SGZ, indicated byarrow in A) of the hippocampus, the region between the granule cell layer (GCL) and hilus (H).

Fig. 3.

Chronic antidepressant treatment increases BrdU labeling in the adult hippocampus. Rats received BrdU injections 4 d after the last ECS or drug treatment, as described in Figure 2. The results are the mean ± SEM number of BrdU-positive cells in hippocampus (n = 8 per group). ECS,Electroconvulsive shock; TCP, tranylcypromine. *p < 0.05 significantly different from vehicle control (F_(3,28) = 7.05;p < 0.05).

Fig. 4.

Chronic, but not acute, fluoxetine administration increases BrdU labeling in the adult hippocampus. Rats were administered fluoxetine for 1, 5, 14, or 28 d and were then given BrdU for analysis of cell proliferation, as described in Figure1. The results are the mean ± SEM number of BrdU-positive cells in hippocampus at each time point (n = 8 per group). *p < 0.05 significantly different from vehicle control (F_(4,35) = 4.35;p < 0.05).

Fig. 5.

Representative photomicrographs of proliferating and mature BrdU-labeled cells. A–C, Photomicrographs of labeled cells 2 hr after BrdU injection (1000×). At this time point proliferating cells are localized to the SGZ and often appear in clusters. The cell clusters in A and Bcontain multiple progenitor cells. Mitotic figures (B,C, indicated by arrow) were also evident in sections from every animal. D, E,Photomicrographs of labeled cells 4 weeks after BrdU injection (1000×). At this time point mature cells are found throughout the granule cell layer and appear ovoid or round, similar to surrounding granule cells. The characteristics of the new and mature cells from control and antidepressant-treated sections were not different. Only the number of proliferating cells was significantly increased by antidepressant treatment.

Fig. 6.

The number of BrdU-positive cells is increased 4 weeks after chronic antidepressant treatment. Rats received BrdU injections 4 d after the last ECS (10 d) or fluoxetine (14 d) treatment and were killed 4 weeks later. The results are the mean ± SEM number of BrdU-positive cells (n = 7 per group). *p < 0.05 significantly different from vehicle control (F_(2,18) = 5.8;p < 0.01).

Fig. 7.

Triple labeling confirms that BrdU-positive cells mature into neurons. Rats received BrdU injections 4 d after the last ECS treatment and were killed 4 weeks later. A representative confocal laser-scanning image (66×) of a section from a fluoxetine-treated rat that has been triple-labeled with BrdU (A, green; BrdU-positive cells indicated byarrows), GFAP (B, blue), and NeuN (C, red). The merged image (D) demonstrates cells that are double-labeled in the GCL for BrdU and NeuN but not GFAP.