Melanocortin-4 receptor regulates hippocampal synaptic plasticity through a protein kinase A-dependent mechanism

Abstract

Learning and memory require orchestrated regulation of both structural and functional synaptic plasticity in the hippocampus. While a neuropeptide alpha-melanocyte-stimulating hormone, α-MSH, has been implicated in memory acquisition and retention, the functional role of its cognate receptor, melanocortin-4 receptor (MC4R), in hippocampal-dependent synaptic plasticity has not been explored. In this study, we report that activation of MC4R enhances synaptic plasticity through the regulation of dendritic spine morphology and abundance of AMPA receptors. We show that activation of postsynaptic MC4R increases the number of mature dendritic spines and enhances surface expression of AMPA receptor subunit GluA1, resulting in synaptic accumulation of GluA1-containing AMPA receptors. Moreover, MC4R stimulates surface GluA1 trafficking through phosphorylation of GluA1 at Ser845 in a Gα(s)-cAMP/PKA-dependent manner. Blockade of protein kinase A (PKA) signaling abolishes the MC4R-mediated enhancement of neurotransmission and hippocampal long-term potentiation. Importantly, in vivo application of MC4R agonists increases LTP in the mouse hippocampal CA1 region. These findings reveal that MC4R in the hippocampus plays a critical role in the regulation of structural and functional plasticity.

Figure 1.

MC4R activation is required for the maintenance of mature spines and functional synapses. a, Expression of MC4R and its ligands in adult mouse hippocampus. mRNAs of different brain parts were prepared and subjected to real time-PCR. The data were presented as relative ratio of mRNA against that of cerebral cortex (agouti was used as a negative control). b, Top row, Staining of surface MC4R in hippocampal neurons at 22 DIV. F-actin was labeled with rhodamine-phalloidin. Scale bar, 5 μm. Bottom row, cultured hippocampal neurons (17 DIV) were transfected with SEP-MC4R and mKOrange constructs, and were examined at 21 DIV. Scale bar, 10 μm. c, d-Tyr MTII increased intracellular cAMP level. Hippocampal neurons were treated with d-Tyr MTII for 30 min (*p < 0.05 versus no treatment, one-way ANOVA with Student-Newman–Keuls test). d, Reduction of MC4R protein in HEK293T cells upon shMC4R knockdown (by ∼83.0 ± 4.2%; ***p < 0.001, n = 3 experiments, Student's t test). The cells were cotransfected with EGFP-tagged rat MC4R expression construct, together with either shMC4R (+) or pSUPER vector (−). e, Cultured hippocampal neurons were cotransfected with shMC4R or its scrambled shRNA (Scr-MC4R) with GFP plasmid. f–h, Hippocampal neurons were cotransfected with shMC4R and constructs expressing shRNA-resistant human MC4R (WT) or its point mutants (D90N or I125K). f, Representative images. g, h, Quantification of spine density (g) and the percentage of mature spines (spines with a mushroom head) (h). Data were expressed as mean ± SEM; **p < 0.01 and ***p < 0.001 versus shMC4R; #p < 0.05 and ### <0.001 versus shMC4R+WT (one-way ANOVA with Student-Newman–Keuls test). i–k, Overexpression of hMC4R-WT and its point mutants (D90N or I250K) in cultured hippocampal neurons. i, Representative images. j, k, Quantification of spine density (j) and percentage of mature spines (k). *p < 0.05 and ***p < 0.001 versus control (Con); ^##p < 0.01 and ^###p <0.001 versus WT (one-way ANOVA with Student-Newman–Keuls test). Scale bars, 10 μm.

Figure 2.

Activation of endogenous MC4R enhances maturation of functional synapses and neurotransmission. a–c, Silencing of MC4R abolished the increase of mature spines and GluA1-containing spines by d-Tyr MTII (1 μm). Hippocampal neurons were cotransfected with GFP plasmid together with shMC4R and mKOrange constructs at 17–18 DIV as indicated. The neurons at 21–22 DIV were treated with d-Tyr MTII for 2 h. a, Representative images. Scale bars: top row, 10 μm; bottom rows, 5 μm. b, c, Quantification of mature spines (b) and SEP-GluA1-containing spines (c). Data were expressed as mean ± SEM; ***p < 0.001, d-Tyr MTII versus control (Con), two-way ANOVA. d–g, MC4R knockdown abolished the d-Tyr MTII-stimulated increase in neurotransmission. Hippocampal neurons were cotransfected with GFP construct together with or without shMC4R and then treated with d-Tyr MTII. d, Representative mEPSC traces. e–g, Cumulative distribution of interevent intervals (inversely proportional to frequency (e), and quantification of frequency (f), and amplitude (g) of mEPSCs. GFP-expressing neurons in the control, the shMC4R-transfected conditions, and their neighboring untransfected neurons (non GFP) were recorded. Data were presented as mean ± SEM; ***p < 0.001, d-Tyr MTII versus Control treatment, two-way ANOVA; ^###p < 0.001 versus GFP cells in shMC4R (with d-Tyr MTII treatment), one-way ANOVA with Student-Newman–Keuls test (f); one-way ANOVA with Kruskal–Wallis test (g).

Figure 3.

MC4R regulates surface levels of GluA1 through PKA-dependent phosphorylation. a–d, Hippocampal neurons were incubated with d-Tyr MTII (100 nm) and H89 (10 μm) for 2 h and then stained for surface GluA1. a, Representative images. Scale bar, 10 μm. b–d, Quantification of density (b), size (c), and intensity (d) of surface GluA1 clusters. Data were expressed as mean ± SEM, **p < 0.01, d-Tyr MTII versus control (two-way ANOVA); n = 10 neurons from each experiment, two experiments). e, Surface and total proteins of hippocampal neurons after d-Tyr MTII treatment were collected and subjected to Western blot analysis for GluA1. f, Fold change (three experiments; *p < 0.05 versus 0 min, one-way ANOVA with Student-Newman–Keuls test. g, h, Hippocampal neurons were cotreated with d-Tyr MTII and H89 for 1 h. g, Western blot analysis. h, Quantitative analysis; *p < 0.05 d-Tyr MTII (+) versus no treatment (−), two-way ANOVA; ^##p < 0.01, versus d-Tyr MTII, one-way ANOVA with Student-Newman–Keuls test. i, d-Tyr MTII increased level of pSer845 GluA1 in cultured hippocampal slices (7 DIV).

Figure 4.

MC4R activation increases basal neurotransmission and LTP induction through PKA activation. a–d, Inhibition of PKA abolished the d-Tyr MTII–stimulated increase in basal neurotransmission. Hippocampal neurons were incubated with d-Tyr MTII (100 nm) and H89 (10 μm) for 2 h. a, Representative mEPSC traces. b–d, Cumulative distribution of interevent intervals (b), quantification of frequency (c), and amplitude (d) of mEPSCs. Data were presented as mean ± SEM, *p < 0.05, d-Tyr MTII versus control (Con), two-way ANOVA (c); ***p < 0.001 versus control, one-way ANOVA with Kruskal–Wallis test (d). e–h, d-Tyr MTII enhanced LTP through activation of PKA. e, f, Basal synaptic transmission and short-term forms of synaptic plasticity were not altered by d-Tyr MTII treatment. e, Input–output curve generated from the slope fEPSP versus fiber volley amplitude. F, Paired pulse facilitation. Second stimuli were delivered at intervals as indicated. Percent facilitation of fEPSP slope of second response as percentage of first response. g, h, Acute hippocampal slices were treated with d-Tyr MTII (1 μm) and H89 (20 μm) for 2 h. g, Summary plot of normalized fEPSP slope measurement (mean ± SEM). h, Quantification of fEPSP slope increase at 1 h after LTP induction (mean ± SEM; **p < 0.01 d-Tyr MTII (+) versus no treatment (−), two-way ANOVA).

Figure 5.

MC4R activation is important for maintaining dendritic spine morphology and enhancement of LTP in vivo. a–c, MC4R knockdown reduced volume of dendritic spines in mouse CA1 hippocampus. a, Representative images. b, Cumulative distribution of dendritic spine volume. Con, Control. c, Length of spines and spine necks. Data were expressed as mean ± SEM; **p < 0.01, ***p < 0.001 (n = 4–5; Student's t test). d, e, Intraperitoneal injection of MC4R agonist d-Tyr MTII or MTII enhanced LTP in mouse CA1 region. d, Plots of normalized fEPSP slope measurement (mean ± SEM). e, Quantification of fEPSP slope increase at 1 h after LTP induction (mean ± SEM; *p < 0.05 versus control; one-way ANOVA with Student-Newman–Keuls test).