Medial PFC AMPA receptor and BDNF signaling are required for the rapid and sustained antidepressant-like effects of 5-HT1A receptor stimulation

Abstract

We previously reported that the serotonergic system is important for the antidepressant-like effects of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist, which produces rapid and long-lasting antidepressant effects in patients with major depressive disorder (MDD). In particular, selective stimulation of the 5-HT_1A receptor in the medial prefrontal cortex (mPFC), as opposed to the somatic 5-HT_1A autoreceptor, has been shown to play a critical role in the antidepressant-like actions of ketamine. However, the detailed mechanisms underlying mPFC 5-HT_1A receptor-mediated antidepressant-like effects are not fully understood. Here we examined the involvement of the glutamate AMPA receptor and brain-derived neurotrophic factor (BDNF) in the antidepressant-like effects of 5-HT_1A receptor activation in the mPFC. The results show that intra-mPFC infusion of the 5-HT_1A receptor agonist 8-OH-DPAT induces rapid and long-lasting antidepressant-like effects in the forced swim, novelty-suppressed feeding, female urine sniffing, and chronic unpredictable stress tests. In addition, the results demonstrate that the antidepressant-like effects of intra-mPFC infusion of 8-OH-DPAT are blocked by co-infusion of an AMPA receptor antagonist or an anti-BDNF neutralizing antibody. In addition, mPFC infusion of 8-OH-DPAT increased the phosphorylation of signaling proteins downstream of BDNF, including mTOR, ERK, 4EBP1, and p70S6K. Finally, selective stimulation of the 5-HT_1A receptor increased levels of synaptic proteins and synaptic function in the mPFC. Collectively, these results indicate that selective stimulation of 5-HT_1A receptor in the mPFC exerts rapid and sustained antidepressant-like effects via activation of AMPA receptor/BDNF/mTOR signaling in mice, which subsequently increase synaptic function in the mPFC, and provide evidence for the 5-HT_1A receptor as a target for the treatment of MDD.

Fig. 1. Infusion of 8-OH-DPAT into the mPFC produces antidepressant-like actions in both unstressed and CUS exposed mice.

a–f Mice were cannulated and after recovery received bilateral infusions of vehicle or 8-OH-DPAT (1, 3 nmol/side), or g–l were exposed to CUS, and then received infusions of 8-OH-DPAT followed by behavioral testing. a–c Location of cannula placement in the mPFC, cannula placements for each animal and experimental time line. d Behavioral testing was conducted 24 h after infusions in the FST (d) and 2 days later in the LMA (Figure S1A). e, f Mice received a second bilateral infusion of 8-OH-DPAT (1, 3 nmol/side) into the mPFC and 24 h later were tested in the 1^stFUST (e), 2 days later in the 2^ndFUST (Figure S1C), and 3 days later in the NSFT (f). Bars represent mean ± SEM ((d): n = 4–8, (e): n = 7, (f): n = 7). ***P < 0.001, **P < 0.01, *P < 0.05 compared with vehicle group, Dunnett’s multiple comparison test, following significant results of one-way ANOVA. g–i Location of cannula placement in the mPFC, cannula placements for each animal and experimental time line. Cannulated mice were exposed to CUS for 14 days, and then received bilateral infusions of vehicle or 8-OH-DPAT (1, 3 nmol/side) 24 h after the last stress exposure. j–l Behavioral testing was conducted in the FST (j) and LMA (Figure S1F) 24 h after 8-OH-DPAT infusions, 2 days later in the SCT (k), and 3 days later in the NSFT (l). Bars represent mean ± SEM ((j): n = 8–13, (k): n = 8–14, (l): n = 8–14). ***P < 0.001, **P < 0.01 compared with vehicle-treated CON group, ^###P < 0.001 compared with vehicle-treated CUS group, Tukey’s multiple comparison test, following significant results of two-way ANOVA [(j–l); (j): interaction, F(1,40) = 2.569, p = 0.1168; (l): interaction, F(1,43) = 4.172, p < 0.05]. Vehicle = saline, DPAT = 8-OH-DPAT, CON = control, CUS = chronic unpredictable stress.

Fig. 2. Pretreatment with an AMPA receptor antagonist blocks the antidepressant-like actions of mPFC-infused 8-OH-DPAT.

a–c Location of cannula placement in the mPFC, cannula placements for each animal and experimental time line. Mice received bilateral infusions of NBQX (0.03 nmol/side) 5 min prior to microinjection of 8-OH-DPAT (3 nmol/side), both into the mPFC. d–f Behavioral testing was conducted in the FST 24 h after 8-OH-DPAT infusions (d), 2 days later to the LMA test (e), and 3 days later in the NSFT (f). Bars represent mean ± SEM; (d): n = 8, (e): n = 8, (f): n = 8. ***P < 0.001, *P < 0.05 compared with vehicle-treated vehicle group, ^##P < 0.01, ^#P < 0.05 compared with vehicle-treated 8-OH-DPAT group, Tukey’s multiple comparison test, following significant results of two-way ANOVA [(d–f); (e): interaction, F(1,28) = 0.6287, p = 0.4345]. Vehicle = saline, DPAT = 8-OH-DPAT.

Fig. 3. Pretreatment with an anti-BDNF nAB blocks the antidepressant-like actions of mPFC-infused 8-OH-DPAT.

a–c Location of cannula placement in the mPFC, cannula placements for each animal and experimental time line. Mice received bilateral infusions of anti-BDNF nAB (0.2 μg/side) 30 min prior to microinjection of 8-OH-DPAT (3 nmol/side), both into the mPFC. d–f Behavioral testing was conducted in the FST 24 h after 8-OH-DPAT infusions (d), 2 days later to the LMA test (e), and 3 days later in the NSFT (f). Bars represent mean ± SEM; (d): n = 7–9, (e): n = 7–9, (f): n = 7–9. ***P < 0.001 compared with vehicle-treated vehicle group, ^###P < 0.001 compared with vehicle-treated 8-OH-DPAT group, Tukey’s multiple comparison test, following significant results of two-way ANOVA [(d–f) (e): interaction, F(1,28) = 0.1282, p = 0.7229]. Vehicle = saline, DPAT = 8-OH-DPAT, BDNF nAB = anti-BDNF nAB.

Fig. 4. Effect of 8-OH-DPAT on downstream of mTORC1 signaling and synaptic proteins in the mPFC.

a Mice received bilateral infusions of 8-OH-DPAT into the mPFC, and 30 min later mPFC was dissected for analysis of phosphoproteins (b) or 24 h later for analysis of synaptic proteins (c). Levels of phosphorylated ERK, 4EBP1, mTOR, and p70S6K, and levels of synapsin 1 and PSD95 were determined by western blot analysis. b Representative images are shown, and density of bands was quantified. The phosphorylation levels of ERK, 4EBP1, mTOR, or p70S6K are presented as a ratio, divided by total ERK, GAPDH, total mTOR, or total p70S6K. c Representative images of synaptic protein western blots are shown. Levels of synapsin 1 and PSD95 are presented as a ratio, divided by GAPDH. d For comparison with mPFC infusions, mice received systemic administration of 8-OH-DPAT (3 mg/kg, s.c.), and mPFC dissections were collected 24 h later. e Representative images of synaptic protein western blots are shown. The levels of Synapsin 1 and PSD95 are presented as a ratio, divided by GAPDH. Bars represent mean ± SEM ((b): n = 7–8, (c): n = 8–10, (e): n = 3–5). *P < 0.05 compared with vehicle group, Student’s t-test. Vehicle = saline, DPAT = 8-OH-DPAT.

Fig. 5. Influence of 8-OH-DPAT infusions into the mPFC on synaptic function in layer V pyramidal neurons.

Mice received bilateral infusions of 8-OH-DPAT (3 nmol/side) into the mPFC 24 h prior to slice preparation and electrophysiological recordings. Layer V pyramidal neurons were patched and 5-HT- or hypocretin-induced EPSCs were determined. a Representative traces of EPSC recordings from vehicle- and 8-OH-DPAT-infused mice. b EPSC frequencies are shown as the mean ± SEM, n = 15–19 cells from six mice. c EPSC amplitudes are mean ± SEM, n = 15–19 cells from six mice. *P < 0.05 compared with vehicle group, Student’s t-test. Vehicle = saline, DPAT = 8-OH-DPAT, Hcrt = hypocretin. d, e During patch clamp recording cells were filled with neurobiotin and then slices were fixed and spine morphology was determined by confocal microscopy. d Representative images of neurobiotin-labeled dendrites are shown. e Infusion of 8-OH-DPAT increased spine head diameter as shown by a shift to the right in the cumulative fraction curves (Kolmogorov–Smirnov two-sample test, D = 0.064, P < 0.0001).