Article Figures & Data
Figures
- Figure 1.
Prolonged epileptiform discharges in the CA3 region of hippocampal slices from Fmr1 knock-out mice after GABAA receptor blockade. A, Intracellular recordings of the spontaneous activity of a CA3 pyramidal cell in a wild-type mouse hippocampal slice after addition of the GABAA receptor antagonist bicuculline (50 μm) to the perfusate. Within 20 min, bicuculline induced rhythmic, short discharges (a) that were ongoing for at least 2 h of continuous recording (b). Here and in B, as well as in Figures 2, A and B, and 3, A and B, the single short discharges illustrated in the main panels are expanded records of the events indicated by the bars in the corresponding insets. These discharges reflected synchronized network activities, because they were recorded at membrane potentials less than –70 mV, they did not change in frequency at different membrane potential levels, and they were recorded with extracellular electrodes (data not shown). Membrane potentials at the beginning of the recordings were as follows: –61 mV (a); –64 mV (b). Burst frequency at 120 min bicuculline appeared greater than that at 20 min. However, no significant change in burst frequency was observed in the overall population (see Results). B, CA3 intracellular recordings from an Fmr1 ko slice after bicuculline addition (50 μm). Bicuculline first induced short synchronized discharges (a) that were similar to those recorded in wild-type slices. Continuous exposure to bicuculline (b), in contrast to what was observed in wild-type experiments, also induced prolonged synchronized discharges of 5–7 s in duration. Membrane potentials were as follows: –62 mV (a); –65 mV (b). C, Time course of the duration of synchronized bursts recorded in the cells shown in A (open circles) and in B (filled circles). The top bar indicates presence of bicuculline in the perfusate. D, Frequency histograms of all synchronized bursts recorded during a 6 min period of stable rhythmic activity, starting at a time point after 80 min of perfusion with bicuculline, in slices from 10 wild-type mice (a) and from 21 Fmr1 ko mice (bi). Note that, in wild-type slices, synchronized discharges were normally distributed with an average duration of 0.588 ± 0.005 s (first-order Gaussian fit; r = 0.91), whereas in Fmr1 ko slices, a two-peak distribution indicate da group of short synchronized discharges with average duration of 0.704 ± 0.007 s and a group of prolonged synchronized discharges of 3.399 ± 0.134 s (second-order Gaussian fit; r = 0.88). bii, Each symbol with a vertical bar represents the average duration and SEM of the five longest synchronized discharges recorded during a 6 min period in each slice from 21 Fmr1 ko animals. In all cases, the duration of the longest synchronized discharges at >80 min of bicuculline perfusion was significantly increased compared with that at 15–30 min bicuculline. Bic, Bicuculline.
- Figure 2.
The mGluR5 antagonist MPEP suppresses the prolonged synchronized discharges in disinhibited Fmr1 knock-out slices. A, Intracellular recordings of CA3 short synchronized discharges in a wild-type slice, after 80 min of perfusion with bicuculline (a) and 60 min after the addition of the mGluR5 antagonist MPEP (50 μm; b) to the perfusate. Membrane potentials were as follows: –65 mV (a); –63 mV (b). B, Intracellular recordings of prolonged epileptiform discharges in CA3 pyramidal cells of an Fmr1 ko slice induced by 80 min of perfusion with bicuculline (a). Sixty minutes after MPEP application, only short synchronized discharges were recorded. Membrane potentials were as follows: –74 mV (a); –73 mV (b). C, Time course of the duration of synchronized bursts recorded in the cells shown in A (open circles) and in B (filled circles). Top bars indicate presence of bicuculline and MPEP in the perfusate. In the Fmr1 ko slice, after 30 min, MPEP reduced the length of the prolonged synchronized discharges to short synchronized discharges similar to those recorded in the wild-type slice. D, Frequency histograms of all synchronized discharges recorded during a 6 min period, at ∼80–120 min of perfusion with bicuculline (top histograms) and at 60–90 min after the addition of MPEP to the perfusate (bottom histograms), in slices from six wild-type mice (a) and from seven Fmr1 ko mice (bi). In wild-type slices, the average burst durations from the peaks of the first-order Gaussian fits were 0.425 ± 0.002 s (r = 0.94) before MPEP and 0.371 ± 0.014 s (r = 0.85) after 60–90 min of MPEP. In Fmr1 ko slices, the two-peak distribution (r = 0.81) before MPEP shows a group of short synchronized discharges of 0.653 ± 0.012 s and a group of prolonged synchronized discharges of 3.015 ± 0.159 s. After 60–90 min of MPEP, the prolonged synchronized discharges were suppressed and a group of short synchronized discharges with a peak at 0.650±0.002 s duration (r = 0.85) remained. bii, Summary plot of the average duration of the five longest synchronized discharges recorded during a 6 min period in slices from seven Fmr1 ko animals at the indicated times. In all experiments, the longest synchronized discharges were prolonged by 80–120 min of bicuculline perfusion and were then significantly reduced in duration by addition of MPEP. Bic, Bicuculline.
- Figure 3.
Inhibitors of mRNA translation suppress induction of prolonged synchronized discharges by DHPG in wild-type slices. A, Effects of the group I mGluR agonist DHPG (50 μm) on the spontaneous activity of CA3 pyramidal cells in wild-type mouse slices that were perfused with ACSF without (a) or with the translation inhibitor anisomycin (20 μm; b) or with the transcription inhibitor actinomycin D (25 μm; c). The inhibitors were present in the perfusing solution for at least 1 h before the addition of DHPG and during perfusion with DHPG. a, As described previously (Zhao et al., 2004), DHPG induced first short synchronized bursts (15 min DHPG) and then prolonged epileptiform discharges (60 min DHPG). b, In the presence of anisomycin, no epileptiform discharges were recorded after 60 min of DHPG (bottom panel). c, In the presence of actinomycin D, epileptiform activity after 60 min of DHPG was similar to that observed in ACSF. Prolonged epileptiform discharges indicated by the bars in a and c, bottom panels, are expanded in d and f, respectively. In the anisomycin-treated slice, addition of bicuculline (50 μm) elicited short synchronized discharges, one of which is shown in e. Membrane potentials (top to bottom): –67, –64, –63 mV (a); –67, –66, –61 mV (b); –66, –63, –67 mV (c); –65 mV (e). B, Summary plot of the average duration of the five longest synchronized discharges recorded during a 6 min period at the indicated time after addition of DHPG in wild-type slices perfused with ACSF (filled circles; n = 8), or treated with actinomycin D (25 μm; filled squares; n = 5), with anisomycin (20 μm; open circles; n = 6), or with cycloheximide (60 μm; open squares). Inhibitors were applied for at least 1 h before the addition of DHPG and were present throughout the experiment. Note that inhibitors of translation, but not of transcription, eventually suppressed the DHPG-induced synchronized discharges. C, Frequency histograms of all synchronized bursts recorded in wild-type slices during a 6 min period, at ∼60–90 min of DHPG application in the absence of actinomycin D (top histogram) and in the presence of actinomycin D (bottom histogram). Second-order Gaussian fits of the events in ACSF (r = 0.80) and in actinomycin D (r = 0.81) show short synchronized discharges (ACSF, 0.376 ± 0.008 s; actinomycin D, 0.400 ± 0.009 s) and prolonged synchronized discharges (ACSF, 4.064 ± 0.111 s; actinomycin D, 4.224 ± 0.038 s) of similar durations.
- Figure 4.
Induction of prolonged synchronized discharges in Fmr1 knock-out slices depends on synaptic activation of group I mGluR-mediated mRNA translation. A, B, Intracellular recordings of the spontaneous activity of CA3 pyramidal cells from a wild-type mouse hippocampal slice (A) and from an Fmr1 ko slice (B), after 20 min (a) and 120 min (b) of bicuculline (50 μm) applied in the presence of an isomycin (20 μm). Before bicuculline application, slices were pretreated with an isomycin for at least 1 h. Membrane potentials were as follows: –66 mV (Aa); –65 mV (Ab); –67 mV (Ba); –69 mV (Bb). C, Intracellular recordings of the spontaneous activity of a CA3 pyramidal cell from an Fmr1 ko slice after 20 min (a) and 120 min (b) of bicuculline (50 μm) applied in the presence of the MEK inhibitor PD98059 (50 μm). Before bicuculline application, the slice was pretreated with PD98059 for ∼1 h. Membrane potentials were as follows: –69 mV (a); –70 mV (b). D, Time course of the duration of the synchronized discharges recorded in the three experiments shown in A (open circles), in B (filled circles), and in C (filled triangles). No prolonged synchronized discharges were observed in anisomycin- or PD98059-treated slices, whereas short synchronized activity occurred apparently unaltered in both wild-type and Fmr1 ko preparations. Additional experiments showed that the mean durations of synchronized discharges after 80–120 min bicuculline in wild-type slices in anisomycin (0.450 ± 0.025 s; n = 7), in Fmr1 ko slices in anisomycin (0.491 ± 0.060 s; n = 8), and in Fmr1 ko slices in PD98059 (0.484 ± 0.039; n = 9) were not significantly different from the duration of synchronized discharges after 80–120 min bicuculline in wild-type slices that were not pretreated with inhibitors of mRNA translation or MEK (0.560 ± 0.055 s; n = 10). E, Summary scheme of FMRP regulation of group I (GpI) mGluR synaptic responses. In the wild-type CA3 area,the FMRP suppression of group I mGluR-mediated responses occurs only at synapses and not at the extra synaptic sites. Without the action of FMRP, as in fragile X preparations, unrestrained mRNA translation triggered by synaptic activation of group I mGluR, via ERK1/2 activation, leads to prolonged synchronized discharges.
