Impaired facilitation of synaptic LTP by R-96544 in Fmr1 knock-out mice

We examined synaptic potentiation in the ACC, which are structures known to be important in learning and fear memory [11], [19]. LTP was induced by pairing presynaptic stimulation with postsynaptic depolarization (see Materials and Methods). The pairing training produced a significant, long-term potentiation of synaptic responses in wild-type (WT) mice (mean, 147.2%±6.5% of baseline; n = 13 slices/5 mice; P<0.01 compared with baseline responses before the pairing training) (Figure 1A). However, the pairing training produced a completely inhibited synaptic potentiation in slices of Fmr1 knock-out (KO) mice (112.7%±9.3%; n = 11 slices/6 mice; P>0.05 compared with baseline responses) (Figure 1A). To detect the effects of 5-HT2A antagonists on the LTP induction, 5-HT2A antagonists were applied during the baseline recording and paring training, after the training, the drugs were washed out. Bath application of the 5-HT2A receptor antagonist, R-96544 (5 μM) or ketanserin (5 μM), for 30 min significantly enhanced the amplitude of LTP (R-96544: 172.8%±6.9%, n = 11 slices/5 mice; ketanserin: 169.8%±6.3%, n = 12 slices/5 mice; P<0.05 versus pairing training only; Figures 1B, C, E, and F). This result suggests that inhibition of 5-HT2AR facilitates LTP induction. However, this facilitation of LTP in WT mice by R-96544 or ketanserin was inhibited in Fmr1 KO mice (R-96544: 108.7%±8.0%, n = 12 slices/5 mice; ketanserin: 105.8%±6.8%, n = 12 slices/5 mice; P>0.05 versus baseline responses Figures 1B, C, E and F). This result demonstrates that serotoninergic modulation of LTP was impaired in Fmr1 KO mice; therefore, FMRP was required for serotoninergic modulation of LTP in the ACC.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. LTP facilitation by R-96544 is through postsynaptic mechanisms.

Paired-pulse facilitation in mice before and after perfusion of R-96544 (the ratio of EPSC2/EPSC1) was recorded with intervals of 35, 50, 75, 100, and 150 ms. (A) Left panel: representative traces of PPF with an interval of 50 ms recorded in the WT and KO mice ACC. Right panel: PPF was similar at each interval in WT and KO mice in the saline control (n = 9 slices/5 mice in each group). Open circles, from WT slice; filled circles, from KO slice. (B) Left panel: representative traces of PPF with intervals of 50 ms recorded in the WT and KO mice. Right panel: PPF was not altered in the WT and KO mice before and after perfusion of R-96544 (n = 10 slices/5 mice in each group). Open circles, from WT slice; filled circles, from KO slice.

https://doi.org/10.1371/journal.pone.0048741.g002

We applied a selective NMDAR antagonist, AP5 (50 μM), to determine whether NMDAR activation was required for the prefrontal LTP induction. The results showed that LTP was completely blocked in the presence of AP5 in WT mice (104.2%±6.3%, n = 9 slices/4 mice; Figure 1D and E). Similarly, LTP was abolished by 10 mM 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetate (BAPTA) in pipette solution (102.6%±6.8%, n = 7 slices/3 mice; Figure 1D and E), indicating that LTP induction depends on the activation of NMDARs and elevation of postsynaptic Ca²⁺ concentrations.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Expression of 5-HT2AR and glutamate receptor subunits.

(A) Representatives of western blot. There were no differences in 5-HT2AR (B), GluR1 (C), NR2A (D), and NR2B (E) expression between the WT and Fmr1 KO mice ACC. n = 4 mice per group.

https://doi.org/10.1371/journal.pone.0048741.g003

No changes of presynaptic glutamate release by blockade of 5-HT2A

In the different regions of the hippocampus, both presynaptic and postsynaptic mechanisms contribute to the expression of LTP [20]. To determine whether presynaptic and/or postsynaptic mechanisms are involved in the serotoninergic modulation of LTP, we measured paired-pulse facilitation (PPF) in the ACC. PPF is a phenomenon by which a second synaptic stimulation of equal magnitude evokes a larger synaptic response than the first; PPF has been used as a tool to implicate the presynaptic probability of transmitter release [21], [22]. Figure 2 shows that no significant difference was observed in PPF induction at five different intervals between WT and Fmr1 KO mice in the presence of R-96544 (5 μM) in the ACC. The data suggest that inhibition of 5-HT2A does not change the presynaptic release during the modulation of LTP induction.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Impaired surface expression of GluR1 by R-96544 in Fmr1 KO neurons.

(A) Representatives of western blot. (B) Surface expression of GluR1 was increased after treatment with 5-HT2AR antagonist R-96544 in the PFC neurons form WT mice (n = 4 dishes). R-96544 failed to trigger GluR1 surface trafficking in the neurons from Fmr1 KO PFC neurons (n = 4 dishes). **P<0.01 compared to control.

https://doi.org/10.1371/journal.pone.0048741.g004

Expression of 5-HT2A and glutamate receptors in the ACC

To further explore the synaptic mechanisms behind the impairment of serotoninergic modulation of LTP, we examined 5-HT2A receptor and glutamate receptor subunits expression in the ACC from WT and Fmr1KO mice. As shown in Figure 3, no differences in the expression of the 5-HT2A receptor were observed between the WT and Fmr1 KO mice as well as in the expression of GluR1 and NMDA receptor subunits NR2A and NR2B under basal conditions (Figures 3A–E). Therefore, the impaired serotoninergic modulation of synaptic potentiation was not caused by a defect in the expression of 5-HT2A and glutamate receptors.

Impaired surface expression of GluR1 stimulated by R-96544 in Fmr1 KO neurons

FMRP acts downstream from the NMDA receptor and contributes to memory-related synaptic potentiation in the ACC of the PFC [7], [11], [12]. To investigate the role of the 5-HT2A receptor signaling pathway in synaptic plasticity, we tested the effects of R-96544 on AMPA receptor GluR1 subunits surface trafficking in cultured PFC neurons in the presence of 1 μM serotonin. The surface expression of GluR1 was increased after treatment with 5-HT2AR antagonist R-96544 (5 μM) (P<0.01 versus control, n = 4 dishes; Figure 4). This finding demonstrates that inhibition of the 5-HT2A receptor can facilitate the surface expression of AMPA GluR1 receptors. However, the surface expression of GluR1 stimulated by R-96544 was impaired in Fmr1 KO PFC neurons (P>0.05, n = 4 dishes; Figure 4). These results indicate that FMRP is involved in serotonin receptor signaling and contributed to GluR1 surface expression induced by 5-HT2A receptor inactivation.

Serotonergic modulation of long-term synaptic plasticity

Synapses in the cingulate cortex undergo plasticity; they are involved in higher brain functions such as learning and memory [12]. Previous studies indicated that 5-HT plays key roles in the maintenance and regulation of balance between potentiation and depression of synaptic networks in the hippocampus [23]. The 5-HT2A receptor antagonist markedly potentiates NMDA response and EPSC in CA1 hippocampus pyramidal cell [17]. Alternatively, the 5-HT2A receptor antagonist can prevent impairment in performance by NMDA receptor blockade in rat PFC [24], [25], [26], [27]. This function is regulated through attenuating the suppression of the NMDA receptor blockade-induced release of cortical glutamate [28]. Therefore, a mutual interaction occurs between serotoninergic and glutamatergic neurotransmissions. In the PFC, including the ACC, activation of the NMDA receptor subunits NR2B and NR2A is critical for the induction of long-term cingulate synaptic plasticity [29], [30]. Previous studies found that the 5-HT2A antagonist enhances the induction of long-term potentiation of CA1 synapses [17]. The present study shows that inhibition of the 5-HT2A receptor facilitates the induction of LTP at ACC synapses. This facilitation of LTP was inhibited in the presence of NMDAR antagonist AP-5 and BAPTA, indicating dependence on the activation of NMDARs and elevated postsynaptic Ca²⁺ concentrations.

FMRP, 5-HT modulation of AMPA receptors, and synaptic potentiation

Our previous study showed that FMRP acts as a key messenger for DA-mediated modulation of excitatory transmission in forebrain neurons [7]. FMRP is required for the surface expression of AMPA GluR1 receptors in response to D1 receptor activation. Facilitation of LTP by D1 receptor activation was consistently absent in the cingulate region of the PFC of Fmr1 KO mice. Recent studies have focused on the interaction between DA and 5-HT receptors. In the intact striatum, studies have consistently demonstrated that intrinsic 5-HT2 receptors modify DA function; furthermore, the divergent roles of 5-HT2A and 5-HT2C receptor subtypes have been postulated [31], [32]. In the present study, we compared the effects of 5-HT2A receptor inhibition on GluR1 surface trafficking in cultured PFC neurons to illustrate the role of FMRP in serotoninergic modulation of GluR1 receptors. We found that inhibition of the 5-HT2A receptor increases GluR1 surface trafficking in ACC neurons; however, this increase was significantly attenuated in Fmr1 KO neurons. This is consistent the electrophysiological recording showing the lost of LTP modulation in the KO nice. Further studies are needed to figure out the molecular signaling involving this disorder.

Several studies proved that the 5-HT2A antagonist can facilitate NMDA receptor-mediated transmission [17], [24], [25], [26], [27], [33]. Moreover, 5-HT2A antagonist significantly increased the amplitude and duration of excitatory postsynaptic potentials and currents evoked by electrical stimulation of the forceps minor [33]. We found that 5-HT2A antagonist facilitates cingulate prefrontal LTP through increased synaptic incorporation of GluR1. The unaltered PPF suggests that the 5-HT2A antagonist does not alter the presynaptic release in the ACC. However, 5-HT reportedly enhances synaptic transmission by increasing the release of glutamate via presynaptic 5-HT2A receptors in rat dorsolateral septal nucleus [34]. The difference between the pre and postsynaptic mechanisms of the 5-HT2A receptor can be attributed to the different distributions of 5-HT receptors in different brain regions. Furthermore, we cannot rule out the possible presynaptic modulation of inhibitory transmission by serotoninergic modulation [35], which was blocked in our recordings.

Animals

For adult brain slice recordings, male mice were 6–8 weeks of age. Fmr1 knock-out mice (strain name: FVB.129P2-Fmr1^tm1Cgr/J; stock #:4624) and control wild-type mice on the same strain background (stock # 4828) were purchased from The Jackson Laboratory (Genetics research, Bar Harbor, Maine, USA) and bred in the animal facility of the Fourth Military Medical University. All mice were housed in plastic boxes in groups of 4 under a 12∶12 light cycle with food and water provided ad libitum. This study was performed in adherence with the National Institutes of Health guidelines for the use of experimental animals. The Institutional Ethical Committee of the Fourth Military Medical University specifically approved this study.

Slice preparation

Coronal brain slices (300 μM) from 6–8-week old Fmr1 WT and KO mice, containing the ACC, were prepared using standard methods [6]. Slices were transferred to submerged recovery chamber with oxygenated (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (ACSF) containing (in mM: 124 NaCl, 4.4 KCl, 2 CaCl₂, 1 MgSO₄, 25 NaHCO₃, 1 NaH₂PO₄, 10 glucose) at room temperature for at least 1 h.

Whole-cell recordings

Experiments were performed in a recording chamber on the stage of an Axioskop 2FS microscope with infrared DIC optics for visualization of whole-cell patch clamp recording. Patch-clamp recordings were performed using Axon 200B amplifiers (Axon Instruments, Union City, CA, USA). In the ACC slices, since layer II-III pyramidal neurons are reported as the major location of intra-cortical horizontal pathways [36] and the superficial layers of the ACC receive substantial glutamate input [37], we recorded EPSCs from neurons in layer II-III induced by the stimulation of layer V. AMPA receptor-mediated EPSCs were induced by repetitive stimulations at 0.02 Hz and neurons were voltage clamped at −70 mV. After obtaining stable EPSCs for at least 10 min, LTP was induced by 80 pulses at 2 Hz paired with postsynaptic depolarization at +30 mV. The recording pipettes (3–5 MΩ were filled with solution containing (mM) 145 K-gluconate, 5 NaCl, 1 MgCl₂, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na₃-GTP (adjusted to pH 7.2 with KOH). Picrotoxin (100 μM) was always present to block GABA_A receptor-mediated inhibitory synaptic currents. Access resistance was 15–30 MΩ and monitored throughout the experiment. Recordings were digitized (10 kHz) on-line using a Digidata 1322 (Axon Instruments) and analyzed off-line with Clamfit 9.0 (Axon Instruments). Data were discarded if access resistance changed more than 15% during an experiment.

Primary culture of prefrontal cortical neurons

The experiments were performed on C57 mice (embryonic, 18 days old of both genders). The Animal Care and Use Committee of the Fourth Military Medical University approved all of the animal protocols used. Cultured prefrontal cortex neurons were prepared as previously described [7]. Briefly, the prefrontal cortex was dissected, minced, and trypsinized for 15 min using 0.125% trypsin (Invitrogen, Carlsbad, USA). The cells were seeded onto 100 mm dishes precoated with 50 μg/ml poly-d-lysine (Sigma) in water and grown in Neurobasal-A medium (Invitrogen) supplemented with B27 and 2 mM GlutaMax (Invitrogen). In the B27/Neurobasal medium, glial growth was reduced to less than 0.5% of the nearly pure neuronal population, as assessed using immunocytochemistry for glial fibrillary acidic protein and neuron specific enolase [38]. The cultures were incubated at 37°C in 95% air/5% carbon dioxide with 95% humidity. The cultures were used for experiments on the 10th day in vitro (DIV 10). No serotonin was added in the experiment; only 5-HT2AR antagonist R-96544 (5 μM, TOCRIS, Bristol, UK) was added in the medium for 6 h before the cells harvested.

Membrane protein preparation

Surface protein samples were separated by superspeed centrifuge. Briefly, cultures were washed three times with ice-cold PBS. Cells were collected and homogenated in buffer A containing 0.32 M sucrose, 5 mM Tris-HCl (PH 7.5), 120 mM KCl, 1 mM EDTA, 0.2 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin A and 1 μg/ml Aprotinin. The lysate was centrifuged at 100,000 g for 1 h at 4°C. Carefully remove and discard the supernatant, the precipitation was obtained and suspended with appropriate buffer B containing 20 mM HEPES (PH 7.5), 10% glycerol, 2% Triton X-100, 1 mM EDTA, 0.2 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin A and 1 μg/ml Aprotinin. After incubating on ice for 2 h, the suspension was centrifuged at 10,000 g for 30 min at 4°C. The majority of membrane protein was in the supernatant.

Western blot

Western blot analysis was performed as described previously [7]. Equal amounts of protein (50 μg) from the cultures were separated and electrotransferred onto PDVF membranes (Invitrogen), which were probed with antibodies for 5-HT2AR (dilution ratio 1∶200, Abcam, Cambridge, MA, #ab66049), GluR1 (dilution ratio 1∶300, Millipore, Billerica, MA, #AB1504), NR2A (dilution ratio 1∶200, Millipore, Billerica, MA, #MAB5216), NR2B (dilution ratio 1∶1000, Millipore, Billerica, MA, #MAB5780), and with β-actin (dilution ratio 1∶10000, Sigma, MO, #A5316) as loading control. For data quantitation, band intensity was expressed relative to the loading control (β-actin). The membranes were incubated with horseradish peroxidase conjugated secondary antibodies (anti-rabbit IgG for the primary antibodies), and bands were visualized using an ECL system (Perkin Elmer).

Data analysis

Results were expressed as mean ± SEM. Statistical comparisons were performed using one- or two-way analysis of variance (ANOVA) using the Student-Newmann-Keuls for post-hoc comparisons. In all cases, P<0.05 was considered statistically significant.