The G Protein-Coupled Serotonin 1A Receptor Augments Protein Kinase Cε-Mediated Neurogenesis in Neonatal Mouse Hippocampus-PKCε-Mediated Signaling in the Early Hippocampus

Abstract

The neurotransmitter serotonin (5-HT) plays an important role in mood disorders. It has been demonstrated that 5-HT signaling through 5-HT_1A receptors (5-HT_1A-R) is crucial for early postnatal hippocampal development and later-life behavior. Although this suggests that 5-HT_1A-R signaling regulates early brain development, the mechanistic underpinnings of this process have remained unclear. Here we show that stimulation of the 5-HT_1A-R at postnatal day 6 (P6) by intrahippocampal infusion of the agonist 8-OH-DPAT (D) causes signaling through protein kinase Cε (PKCε) and extracellular receptor activated kinase ½ (ERK1/2) to boost neuroblast proliferation in the dentate gyrus (DG), as displayed by an increase in bromodeoxy-uridine (BrdU), doublecortin (DCX) double-positive cells. This boost in neuroproliferation was eliminated in mice treated with D in the presence of a 5-HT_1A-R antagonist (WAY100635), a selective PKCε inhibitor, or an ERK1/2-kinase (MEK) inhibitor (U0126). It is believed that hippocampal neuro-progenitors undergoing neonatal proliferation subsequently become postmitotic and enter the synaptogenesis phase. Double-staining with antibodies against bromodeoxyuridine (BrdU) and neuronal nuclear protein (NeuN) confirmed that 5-HT_1A-R → PKCε → ERK1/2-mediated boosted neuroproliferation at P6 also leads to an increase in BrdU-labeled granular neurons at P36. This 5-HT_1A-R-mediated increase in mature neurons was unlikely due to suppressed apoptosis, because terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis showed no difference in DNA terminal labeling between vehicle and 8-OH-DPAT-infused mice. Therefore, 5-HT_1A-R signaling through PKCε may play an important role in micro-neurogenesis in the DG at P6, following which many of these new-born neuroprogenitors develop into mature neurons.

Keywords: 5-HT1A receptor; PKC isozymes; dentate gyrus; neonatal; neurogenesis.

Figure 1

Serotonin 1A receptor-mediated activation of PKCε and ERK1/2 in proliferating hippocampal neuron-derived HN2-5 cells. (a) Relative to carrier (C) (vehicle) treatment, agonist (8-OH-DPAT, D) (100 nM) caused maximal activation of PKCε in 20 min (measured using a P-Ser⁷²⁹-PKCε antibody and normalized to ERK), which was eliminated in the presence of the 5-HT_1A-R antagonist WAY100635 (WAY) (10 µM), but not in the presence of the MEK inhibitor U0126 (U) (10 µM). (b) Relative to carrier treatment (C), 8-OH-DPAT (D) treatment (100 nM) caused a dramatic increase in the activity of ERK1/2 in 30 min (measured using a P-T²⁰², Y²⁰⁴-ERK antibody, normalized to ERK), and this activation was blocked in the presence of the PKCε inhibitor (M) (400 nM) and also U0126 (10 µM). In (b) * p < 0.05, D versus carrier and each of the inhibitors (t-test). Data were analyzed from distinct Western blots obtained from three different cultures of HN2-5 cells. (See Figure S1 in Supplementary Material for the effect of inhibitors alone).

Figure 2

Developmental Profile of the G-protein-coupled form of the 5-HT_1A-R in the hippocampus. Hippocampi culled from each mouse were pooled and processed as one sample. [³H] 8-OH-DPAT binding assay was conducted using 250 μg hippocampal membrane protein per tube for each developmental stage. Membrane samples were analyzed in triplicate tubes and the experiment was conducted on three mice (n = 3) for each developmental time point. Data obtained were plotted with standard deviations. The sharp increases between P4 and P6 as well as between P10 and P15 were statistically significant (t-test; p < 0.05).

Figure 3

Stimulated 5-HT_1A-R causes PKCε-mediated activation of ERK1/2 in P6 DG. One hour after intra-hippocampal infusion of drugs, the hippocampi from P6 pups were processed for P-T²⁰², Y²⁰⁴-ERK (P-ERK) (red), NeuN (green) double staining. (a) 8-OH-DPAT (D) (100 nM) treatment caused an increase in nuclear P-ERK, which was suppressed in the presence of the MEK inhibitor U0126 (U), and also a selective PKCε inhibitor (Myr-εV1-2) (M). (b) Quantification of the number of P-ERK (+) nuclei was performed from three sections (every sixth) from each of the dorsal, dorso-ventral, and ventral regions (nine sections in total per mouse pup). Three to four mice per treatment group were used (see results for details). The count of P-ERK (+) nuclei within the DG (in the dotted contour) for each section was normalized to the count of NeuN (+) nuclei and then the normalized mean from these nine sections was used for each mouse to conduct statistical analysis. One-way ANOVA (F(4,12) = 117.45, p < 0.0001) and Scheffé for post-hoc tests yielded p < 0.05 for D compared to the other groups (inset). Note: M, U, WAY (W) block D-evoked activation of ERK (activation marked by nuclear translocation of P-ERK). Scale bar: 47.62 µm.

Figure 4

Stimulated 5-HT_1A-R elicits PKCε and ERK1/2-mediated neuroproliferation in P6 DG. C57BL/6 P6 pups were injected (i.p.) with BrdU 2 h before intra-hippocampal infusion of D (100 nM) in the absence or presence of the inhibitors. (a–e) Number of neuroblasts proliferating in P6 DG 24 h after intra-hippocampal infusion of carrier (vehicle) or D in the absence (b) and presence of W (c), M (d) and U (e). (b) (enlarged) Neuroproliferation was shown by the co-localization of BrdU (red) and DCX (blue) yielding a pink–purple color. (f) A diagrammatic representation of the 5-HT_1A receptor-signaling cascade highlighting the hierarchy of the key players PKCε and MAPK/ERK. (g) Mean BrdU, DCX (++) cell number in the hippocampal DG per section from nine sections per mouse pup was used for statistical analysis. We used three mice per treatment (n = 3) (see results for details); (One-way ANOVA: F(4,10) = 67.17, p < 0.0001). Post-hoc analysis showed p < 0.05 for D compared to the other groups (inset). Scale bar: 75 µm. (h) Enlarged image of a BrdU, DCX (++) cell showing BrdU localization in the nucleus and DCX staining in the cytoplasm.

Figure 5

Activated 5-HT_1A-R evokes PKCε and ERK1/2-mediated neurogenesis in P6 DG. After BrdU treatment (i.p.), one set of five pups in each experiment was dedicated to fluoxetine (Flx) infusion alone (Figure S2). Two such experiments were conducted to obtain the results presented here. Thirty days after treatment and intrahippocampal infusion of D in the absence or presence of inhibitors, the brains were processed for immunohistochemistry (IHC). (a–f) Co-localization of BrdU (red) with NeuN (green) in cells (shown using white arrows) indicated increased neurogenesis with D, which was eliminated in the presence of antagonist (W) (c), M (d), and U (e). Lower panels: red BrdU (+) cells shown by blocking the green and blue channels. (f) Mean BrdU, NeuN (++) cell number in the DG per hippocampal section calculated from nine sections per mouse and four or five mouse pups per treatment showed that the D group harbored significantly higher BrdU, NeuN (++) cells than all other groups (see Results for details and Figure S2 for the Flx set). (One-way ANOVA: F(5,21) = 36.47, p < 0.0001) and post-hoc tests showed that the D group was significantly higher (p < 0.05) than the inhibitor-treated groups. (g) Treatment strategy and time line. Scale bar: 75 µm. * p < 0.05.

Figure 5

Activated 5-HT_1A-R evokes PKCε and ERK1/2-mediated neurogenesis in P6 DG. After BrdU treatment (i.p.), one set of five pups in each experiment was dedicated to fluoxetine (Flx) infusion alone (Figure S2). Two such experiments were conducted to obtain the results presented here. Thirty days after treatment and intrahippocampal infusion of D in the absence or presence of inhibitors, the brains were processed for immunohistochemistry (IHC). (a–f) Co-localization of BrdU (red) with NeuN (green) in cells (shown using white arrows) indicated increased neurogenesis with D, which was eliminated in the presence of antagonist (W) (c), M (d), and U (e). Lower panels: red BrdU (+) cells shown by blocking the green and blue channels. (f) Mean BrdU, NeuN (++) cell number in the DG per hippocampal section calculated from nine sections per mouse and four or five mouse pups per treatment showed that the D group harbored significantly higher BrdU, NeuN (++) cells than all other groups (see Results for details and Figure S2 for the Flx set). (One-way ANOVA: F(5,21) = 36.47, p < 0.0001) and post-hoc tests showed that the D group was significantly higher (p < 0.05) than the inhibitor-treated groups. (g) Treatment strategy and time line. Scale bar: 75 µm. * p < 0.05.

Figure 6

TUNEL assays reveal that 8-OH-DPAT treatment in vivo does not block apoptosis in the DG. TUNEL (+) cells were marked green within the HOECHST33342-stained dentate gyrus granule cells. TUNEL assays were performed with nine sections per brain from four mouse pups per treatment (n = 4). The groups of mice were treated with (a) carrier (vehicle) or 8-OH-DPAT (D) for 24 h (to analyze apoptosis during neuroproliferation) or (b) 30 days (to analyze apoptosis during neurogenesis). (c,f) Neither experiment recorded a significant decrease in TUNEL (+) cells in the DG. (c,f) A large number of TUNEL (+) apoptotic cells were observed in the subventricular zone (SVZ) (white arrows) of the carrier (vehicle)-treated but not in the 8-OH-DPAT-treated mice (d,e). Student’s t-test was used for data analysis. Scale bar: 47.62 µm.

Figure 6

TUNEL assays reveal that 8-OH-DPAT treatment in vivo does not block apoptosis in the DG. TUNEL (+) cells were marked green within the HOECHST33342-stained dentate gyrus granule cells. TUNEL assays were performed with nine sections per brain from four mouse pups per treatment (n = 4). The groups of mice were treated with (a) carrier (vehicle) or 8-OH-DPAT (D) for 24 h (to analyze apoptosis during neuroproliferation) or (b) 30 days (to analyze apoptosis during neurogenesis). (c,f) Neither experiment recorded a significant decrease in TUNEL (+) cells in the DG. (c,f) A large number of TUNEL (+) apoptotic cells were observed in the subventricular zone (SVZ) (white arrows) of the carrier (vehicle)-treated but not in the 8-OH-DPAT-treated mice (d,e). Student’s t-test was used for data analysis. Scale bar: 47.62 µm.