Activation of Serotonin 5-HT7 Receptors Modulates Hippocampal Synaptic Plasticity by Stimulation of Adenylate Cyclases and Rescues Learning and Behavior in a Mouse Model of Fragile X Syndrome

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.

Keywords: 5-HT7 receptor; PACAP; cyclic AMP; fragile X syndrome; learning; mGluR-LTD; serotonin.

Figure 1

Modulation of adenylate cyclase activity modified metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in wild-type (WT) and Fmr1 Knockout (KO) slices. AMPAR-mediated excitatory post-synaptic currents (EPSCs) were recorded in the presence of D-AP5 (50 μM) and bicuculline (5 μM) under whole-cell patch clamp in the CA3–CA1 synapse in hippocampal slices from WT and Fmr1 KO mice. (A) In WT slices, bath application of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μM, 5 min) induced a long-term depression (mGluR-LTD) of EPSC amplitude (white dots, n = 11). When the adenylate cyclase blocker SQ 22536 (SQ, 10 μM) was added to intracellular medium, DHPG-induced mGluR-LTD was slightly enhanced (gray dots, n = 8) with respect to control. Individual representative EPSC traces are shown on top of the graph (1: baseline; 2: acute EPSC reduction; 3: mGluR-LTD). (B) In Fmr1 KO slices, DHPG-induced mGluR-LTD (black dots, n = 9) was enhanced with respect to WT (A, white dots) and was not further enhanced in the presence of intracellular SQ 22536 (SQ, 10 μM; dark gray dots, n = 6). (C) The bar graph shows the amount of mGluR-LTD (mean EPSC amplitude in all tested neurons, expressed as % of baseline EPSC amplitude). The amount of mGluR-LTD was compared by one-way ANOVA followed by Tukey’s multiple comparisons test in the four experimental conditions illustrated in (A,B) (*P = 0.03). In control conditions (DHPG), mGluR-LTD in Fmr1 KO was significantly enhanced with respect to WT (*P = 0.025, by unpaired t-test). In the presence of intracellular SQ 22536 (DHPG+SQ), the amount of mGluR-LTD in WT was not significantly different from Fmr1 KO (P = 0.83, by unpaired t-test) and was also comparable to that observed in Fmr1 KO in control conditions (P = 0.35 by unpaired t-test). (D) When DHPG application was followed by bath application of forskolin, a direct activator of adenylate cyclase (20 μM, 5 min), mGluR-LTD was reduced (gray dots, n = 4) with respect to control conditions (white dots; n = 5). (E) The same result was observed in Fmr1 KO slices: DHPG-induced mGluR-LTD (white dots; n = 9) was completely reversed by application of forskolin (20 μM, 5 min; gray dots, n = 5). (F) Reversal of mGluR-LTD by forskolin was statistically significant both in WT (*P = 0.025; by unpaired t-test) and in Fmr1 KO (**P = 0.0026).

Figure 2

Pituitary adenylate cyclase activating peptide (PACAP) reversed mGluR-LTD in WT and in Fmr1 KO hippocampus. mGluR-LTD was induced by application of DHPG (100 μM, 5 min). Application of PACAP (10 nM, 5 min) fully reversed DHPG-induced mGluR-LTD in WT (A) and in Fmr1 KO slices (C). (B,D) Reversal of mGluR-LTD by PACAP was statistically significant both in WT (*P = 0.04, by unpaired t-test) and in Fmr1 KO slices (**P = 0.0025, unpaired t-test).

Figure 3

Activation of 5-HT₇ receptors reversed mGluR-LTD by stimulation of adenylate cyclase. (A) In hippocampal WT slices, application of LP-211 did not modify mGluR-LTD in the presence of the adenylate cyclase blocker SQ 22536 (10 μM, included in intracellular pipette solution; dark gray dots, n = 6). (B) The bar graph shows the amount of mGluR-LTD measured 45 min after LTD induction (mean EPSC amplitude in all tested neurons, expressed as % of baseline EPSC amplitude). In WT slices, bath applications of DHPG-induced mGluR-LTD (white column; n = 11) that was completely reversed when DHPG application was followed by application of the 5-HT₇R agonist LP-211 (10 nM, 5 min; black column, n = 7; *P = 0.03 by unpaired t-test). In the presence of intracellular SQ 22536 (10 μM; gray column, n = 6), the amount of mGluR-LTD was slightly increased with respect to control and was not reversed by application of LP-211 (10 nM, 5 min; dark gray column, n = 6; significantly different from the effect of LP-211 in control conditions, **P = 0.0026 by unpaired t-test). (C) Similarly, in Fmr1 KO slices application of LP-211 (10 nM, 5 min) had no effect on mGluR-LTD in the presence of intracellular SQ 22536 (10 μM; dark gray column, n = 6). (D) In Fmr1 KO slices, application of LP-211 reversed DHPG-induced mGluR-LTD only in control conditions (black column, n = 6; *P = 0.02 by unpaired t-test) but not in the presence of SQ 22536 (SQ, 10 μM; gray column, n = 6), showing that 5-HT₇R-mediated effect required stimulation of adenylate cyclase also in Fmr1 KO slices.

Figure 4

5-HT₇ receptor-mediated reversal of mGluR-LTD required protein kinase A (PKA) and was occluded by inhibition of extracellular signal regulated kinase (ERK). mGluR-LTD was induced by bath application of DHPG (100 μM, 5 min) in WT slices in control conditions and in the presence of peptide fragment 6–22 (PKI, added to intracellular solution), an inhibitor of PKA. (A) Application of LP-211 (10 nM, 5 min) reversed mGluR-LTD in control conditions (black dots, n = 7) but had no effect in the presence of PKI (dark gray dots, n = 5). (B) In the presence of PKI, the effect of LP-211 was significantly reduced (**P = 0.0013, by unpaired t-test), indicating that PKA activation was necessary for 5-HT₇R-mediated reversal of mGluR-LTD. (C) mGluR-LTD was induced by bath application of DHPG (100 μM, 5 min) in WT slices in control conditions (white dots, n = 11) and in the presence of the mitogen-activated protein kinase (MAPK)/ERK blocker PD-98059 (PD, 40 μM, added to intracellular solution). Intracellular PD-98059 completely reversed mGluR-LTD (dark gray dots, n = 5) and occluded the effect of LP-211 (10 nM, 5 min; gray dots, n = 5). (D) mGluR-LTD was significantly reversed by PD-98059 (*P = 0.03) and was not further modified by LP-211 (P = 0.52).

Figure 5

Activation of 5-HT₇ receptors stimulated ERK phosphorylation. (A–C) Representative immunoblots showing the levels of phosphorylated and total ERK1/2 in control and LP-211 treated (10 nM, 5 min) hippocampal slices from WT (FVB strain; A), WT (C57BL/6J strain; B), and Fmr1 KO (C57BL/6J strain; C) mice. (D–F) Semi-quantitative analysis of phosphorylated ERK1/2 vs. total ERK1/2 in control and LP-211 treated hippocampal slices from WT (FVB strain; D), WT (C57BL/6J strain; E), and Fmr1 KO (C57BL/6J; F) mice. GAPDH was used as loading control. Relative optical density is presented as percentage of control. Data represent mean ± SEM of five (D), four (E,F) separate experiments, each performed on a pool of three mice. **p = 0.0064 by unpaired t-test. Full length immunoblots are shown in Supplementary Figures S2, S3.

Figure 6

Acute in vivo administration of LP-211 improved memory and reduced stereotyped behavior in Fmr1 KO mice. (A) Exploration times of familiar and novel object during T2 (after a 24-h retention interval) show that Fmr1 KO mice treated with vehicle present an impairment of memory (higher amount of time exploring the familiar vs. the novel object; p < 0.0001) that is rescued by treatment with LP-211 (3 mg/kg 30 min before T1). WT + vehicle = 11; Fmr1 KO + vehicle = 12; WT + LP-211 = 10; Fmr1 KO + LP-211 = 10. (B) Analysis of the discrimination (D) index confirms that the impairment of recognition memory in Fmr1 KO (P = 0.015 vs. WT) is rescued by LP-211 (P = 0.02 vs. Fmr1 KO + LP-211). A difference from 0 is depicted with hashes (^#p < 0.05) (C) Latency to first approach to the novel object and (D) total exploration time are comparable in the 4 groups of mice. (E) Fmr1 KO treated with vehicle buried a higher number of marbles compared to WT (P = 0.002). This stereotypic behavior was rescued by treatment with LP-211 (3 mg/kg 30 min before test). WT + vehicle = 11; WT + LP-211 = 10; Fmr1 KO + vehicle = 12; Fmr1 KO + LP-211 = 10 for novel object recognition (NOR; A–D) and Marble Burying (E) tasks. (F) Time spent in the center of the arena and (G) the number of crosses into the center is comparable in the four groups of mice. (H–I) General locomotor activity analyzed as horizontal activity and vertical activity (rearing) is not modified by genotype and treatment. (J) The increase in stereotyped behavior such as grooming in Fmr1 KO mice (P < 0.0001) is rescued by treatment with LP-211. (K) Fmr1 KO mice show a decrease of spontaneous freezing behavior compared to WT littermates (P = 0.036) that is rescued by LP-211. (L) Defecation is comparable in the four groups of mice. WT + vehicle = 10; WT + LP-211 = 12; Fmr1 KO + vehicle = 12; Fmr1 KO + LP-211 = 14 for Open field (OF) test. Data are expressed as mean ± SEM. *Significant difference (p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test); ^#difference with zero (one-sample t-test).