Reversal of hippocampal neuronal maturation by serotonergic antidepressants

Abstract

Serotonergic antidepressant drugs have been commonly used to treat mood and anxiety disorders, and increasing evidence suggests potential use of these drugs beyond current antidepressant therapeutics. Facilitation of adult neurogenesis in the hippocampal dentate gyrus has been suggested to be a candidate mechanism of action of antidepressant drugs, but this mechanism may be only one of the broad effects of antidepressants. Here we show a distinct unique action of the serotonergic antidepressant fluoxetine in transforming the phenotype of mature dentate granule cells. Chronic treatments of adult mice with fluoxetine strongly reduced expression of the mature granule cell marker calbindin. The fluoxetine treatment induced active somatic membrane properties resembling immature granule cells and markedly reduced synaptic facilitation that characterizes the mature dentate-to-CA3 signal transmission. These changes cannot be explained simply by an increase in newly generated immature neurons, but best characterized as "dematuration" of mature granule cells. This granule cell dematuration developed along with increases in the efficacy of serotonin in 5-HT(4) receptor-dependent neuromodulation and was attenuated in mice lacking the 5-HT(4) receptor. Our results suggest that serotonergic antidepressants can reverse the established state of neuronal maturation in the adult hippocampus, and up-regulation of 5-HT(4) receptor-mediated signaling may play a critical role in this distinct action of antidepressants. Such reversal of neuronal maturation could affect proper functioning of the mature hippocampal circuit, but may also cause some beneficial effects by reinstating neuronal functions that are lost during development.

Fig. 1.

Chronic fluoxetine down-regulates markers for mature granule cells. (A) Reduction in calbindin-like immunoreactivity, increase in calretinin-positive cells, but no appreciable change in NeuN-like immunoreactivity or DAPI staining in DG of fluoxetine-treated mice (FLX) compared with control mice (CNT). (Scale bar, 100 μm.) (B) Reduced calbindin protein levels in fluoxetine-treated DG revealed by immunoblot analyses (n = 5 each, P = 0.0079). (C) Reduced expression levels of mRNAs for calbindin, desmoplakin (Dsp), tryptophan-2,3-dioxygenase (TDO), and type I interleukin 1 receptor (IL1R) in fluoxetine-treated DG (n = 6 each). **, P < 0.01; ***, P < 0.005. (D) Confocal images showing immunostaining for BrdU (green) and calbindin (red) in GC layer. Images were from 9-week-old mice before fluoxetine treatment (Left), 13-week-old control (Center), and 13-week-old fluoxetine-treated mice (Right). Arrow: BrdU-positive cells without calbindin-like immunoreactivity. Arrow head: cells with calbindin-like immunoreactivity. (Scale bar, 10 μm.) Data are presented as mean ± SEM.

Fig. 2.

Dentate granule cells in fluoxetine-treated mice show immature-like natures. (A) Left, c-Fos-like immunoreactivity in GC layer after foot shocks. Right, quantitative data showing the number of c-Fos-positive cells in mice in home cages and after foot shocks (n = 4 each). Fluoxetine strongly reduced foot shock-induced c-Fos expression (P = 0.0286). (Scale bar, 200 μm.) (B) Sample recordings of GC spikes (Upper) evoked by depolarizing currents (Lower). (Scale bars: 100 ms, 40 pA, and 50 mV.) (C) Left, the minimal current intensity required to evoke a single spike is smaller in fluoxetine-treated GCs (CNT, n = 31 cells; FLX, n = 35 cells; P = 0.0099). Right, no significant difference in input resistance. (D) Left, TTX (1 μM) completely blocked spikes in control cells. Right, TTX-resistant component (arrow) in fluoxetine-treated cells was blocked by Ni²⁺ (50 μM). The magnitude of injected currents was 140 pA (CNT) and 100 pA (FLX). (Scale bars: 50 ms, 50 mV (Top), and 20 mV (Middle and Bottom). Data are presented as mean ± SEM.

Fig. 3.

Chronic fluoxetine reduces mossy fiber synaptic facilitation to juvenile level. (A) Pooled data showing marked reduction of 1 Hz frequency facilitation at the MF synapse in fluoxetine-treated mice (CNT, n = 30; FLX, n = 35; see Fig. S5A for statistics). Sample recordings are averages of 15 consecutive fEPSPs during baseline and 1 Hz stimulation. (Scale bars: 10 ms and 0.5 mV.) (B) A histogram of magnitude of facilitation at 1 Hz. (C) Reduced frequency facilitation at 0.2 Hz (n = 14 each, P < 0.0001) and 100 Hz (n = 8 each, P = 0.0022). (D) No significant difference in ratios of fEPSP to fiber volley amplitude (CNT, n = 18; FLX, n = 20). (E) Developmental increases in magnitude of 1 Hz facilitation. Each symbol represents a single mouse. The dotted line shows the median value in adult fluoxetine-treated mice. (F) Higher GC excitability in mice with smaller synaptic facilitation. Fluoxetine-treated mice were divided into two groups (five mice each) by order of magnitude of 1 Hz facilitation, and the threshold current for GC spike generation was compared (large, n = 19 cells; small, n = 16 cells; P = 0.0093). Data are presented as mean ± SEM.

Fig. 4.

Involvement of 5-HT₄ receptor in effects of chronic fluoxetine. (A) Fluoxetine at 22 mg/kg/day augmented MF synaptic potentiation induced by bath application of 5-HT for 5 min (CNT, n = 9; FLX, n = 9; P = 0.004). Sample recordings are averages of nine consecutive fEPSPs during baseline and 5-HT application. (Scale bars: 10 ms and 0.2 mV.) (B) Dose-dependent effects of fluoxetine on 5-HT-induced potentiation. Each symbol represents a single mouse. In mice shown by triangles, fluoxetine concentrations were calculated on the basis of averaged water consumption, as in Fig. S5B. The dotted line shows the control level. (C) Correlation between frequency facilitation and 5-HT-induced potentiation. Data from mice treated at 18–22 mg/kg/day were included. P < 0.0001, r² = 0.6657. (D) Lack of 5-HT-induced synaptic potentiation in 5-HT₄ receptor-deficient mice (^+/+, n = 6; ^−/−, n = 9). (E) Significant effects of chronic fluoxetine on calbindin levels in wild-type (n = 5 each, P = 0.0159), but not in mutant mice (n = 6 each). (F) Significant effects of fluoxetine on frequency facilitation in wild-type (CNT, n = 6; FLX, n = 5; P = 0.0173), but not in mutant mice (CNT, n = 9; FLX, n = 7). Data are presented as mean ± SEM.