Abstract
We have previously demonstrated that activation of serotonin 5-HT7 receptors (5-HT7R) reverses metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in the hippocampus of wild-type (WT) and Fmr1 Knockout (KO) mice, a model of Fragile X Syndrome (FXS) in which mGluR-LTD is abnormally enhanced. Here, we have investigated intracellular mechanisms underlying the effect of 5-HT7R activation using patch clamp on hippocampal slices. Furthermore, we have tested whether in vivo administration of LP-211, a selective 5-HT7R agonist, can rescue learning and behavior in Fmr1 KO mice. In the presence of an adenylate cyclase blocker, mGluR-LTD was slightly enhanced in WT and therefore the difference between mGluR-LTD in WT and Fmr1 KO slices was no longer present. Conversely, activation of adenylate cyclase by either forskolin or Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) completely reversed mGluR-LTD in WT and Fmr1 KO. 5-HT7R activation reversed mGluR-LTD in WT and corrected exaggerated mGluR-LTD in Fmr1 KO; this effect was abolished by blockade of either adenylate cyclase or protein kinase A (PKA). Exposure of hippocampal slices to LP-211 caused an increased phosphorylation of extracellular signal regulated kinase (ERK), an intracellular effector involved in mGluR-LTD, in WT mice. Conversely, this effect was barely detectable in Fmr1 KO mice, suggesting that 5-HT7R-mediated reversal of mGluR-LTD does not require ERK stimulation. Finally, an acute in vivo administration of LP-211 improved novel object recognition (NOR) performance in WT and Fmr1 KO mice and reduced stereotyped behavior in Fmr1 KO mice. Our results indicate that mGluR-LTD in WT and Fmr1 KO slices is bidirectionally modulated in conditions of either reduced or enhanced cAMP formation. Activation of 5-HT7 receptors reverses mGluR-LTD by activation of the cAMP/PKA intracellular pathway. Importantly, a systemic administration of a 5-HT7R agonist to Fmr1 KO mice corrected learning deficits and repetitive behavior. We suggest that selective 5-HT7R agonists might become novel pharmacological tools for FXS therapy.
Highlights
Fragile X Syndrome (FXS), the most common single-gene cause of intellectual disability, autism and epilepsy (Garber et al, 2008), is caused by transcriptional silencing of the FMR1 gene coding for Fragile X Mental Retardation Protein (FMRP), an mRNA-binding protein that regulates translation of several synaptic proteins (Pfeiffer and Huber, 2009)
Excitatory post-synaptic currents (EPSCs) mediated by AMPA receptors were recorded in the CA3-CA1 synapse on hippocampal slices from WT and Fmr1 KO mice. metabotropic glutamate receptors (mGluRs)-LTD of excitatory post synaptic currents (EPSCs) was chemically induced by bath application of the mGluR agonist DHPG (100 μM, 5 min)
When the adenylate cyclase inhibitor SQ 22536 (10 μM) was included in the intracellular pipette solution (Figure 1A), the amount of mGluR-LTD in WT slices showed a trend towards an enhancement compared to control conditions, not statistically significant (EPSC % amplitude measured 45 min after LTD induction: 79.5 ± 10, vs. 56 ± 9 comparing control vs. SQ 22536, n = 11/7; unpaired t-test: t(16) = 1.56; P = 0.07)
Summary
Fragile X Syndrome (FXS), the most common single-gene cause of intellectual disability, autism and epilepsy (Garber et al, 2008), is caused by transcriptional silencing of the FMR1 gene coding for Fragile X Mental Retardation Protein (FMRP), an mRNA-binding protein that regulates translation of several synaptic proteins (Pfeiffer and Huber, 2009). Studies on the mouse model of FXS revealed altered synaptic plasticity mediated by metabotropic glutamate receptors (mGluRs; Pfeiffer and Huber, 2009). Metabotropic glutamate receptor-mediated long term depression (mGluR-LTD), a form of plasticity playing a crucial role in cognition and in behavioral flexibility (Luscher and Huber, 2010; Sanderson et al, 2016), is pathologically enhanced in the hippocampus of Fmr KO mice (Huber et al, 2002) and is regarded as the electrophysiological readout of synaptic malfunction in the mouse model of FXS (Bear et al, 2004; Waung and Huber, 2009). Reduced cAMP production was later detected in the brain of dfmr null drosophila, in brain and blood platelets of Fmr KO mice and in neural precursor cells from human FXS fetal tissues (Kelley et al, 2007). Pharmacological manipulation with agents that potentially increase cAMP, i.e., inhibitors of group II mGluRs and phosphodiesterase IV inhibitors (PDE4-Is), reversed the mGluR-LTD alteration in FXS mouse models (Choi et al, 2011, 2015, 2016), leading to the hypothesis that increasing cAMP formation might become a potential therapeutic strategy to rescue FXS phenotype
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