Synaptic ionotropic glutamate receptors and plasticity are developmentally altered in the CA1 field of Fmr1 knockout mice - PubMed (original) (raw)

Comparative Study

Synaptic ionotropic glutamate receptors and plasticity are developmentally altered in the CA1 field of Fmr1 knockout mice

Yair Pilpel et al. J Physiol. 2009.

Abstract

Fragile X syndrome is one of the most common forms of mental retardation, yet little is known about the physiological mechanisms causing the disease. In this study, we probed the ionotropic glutamate receptor content in synapses of hippocampal CA1 pyramidal neurons in a mouse model for fragile X (Fmr1 KO2). We found that Fmr1 KO2 mice display a significantly lower AMPA to NMDA ratio than wild-type mice at 2 weeks of postnatal development but not at 6-7 weeks of age. This ratio difference at 2 weeks postnatally is caused by down-regulation of the AMPA and up-regulation of the NMDA receptor components. In correlation with these changes, the induction of NMDA receptor-dependent long-term potentiation following a low-frequency pairing protocol is increased in Fmr1 KO2 mice at this developmental stage but not later in maturation. We propose that ionotropic glutamate receptors, as well as potentiation, are altered at a critical time point for hippocampal network development, causing long-term changes. Associated learning and memory deficits would contribute to the fragile X mental retardation phenotype.

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Figures

Figure 2

Figure 2. AMPA/NMDA ratios are altered at P14, but not at 6–7 weeks

A and B, scaled sample traces of AMPA/NMDA ratio measurements at P14 and 6–7 weeks, respectively. Black traces are from WT control cells and grey traces are from Fmr1 KO2 littermates. In all cases, in order to afford easy comparison of the ratio between the AMPA and the NMDA responses, the current at a holding potential of −90 mV was scaled to the level of peak current flow at a +40 mV holding potential in the same recording. Numbers on the _X_-axis denote time after stimulation. Note that AMPA currents (negative current traces) are virtually gone 50 ms after stimulation (as seen by the stimulus artifact). In A, a vertical line has been drawn to denote where NMDA current values are measured (NMDA). The AMPA current values are measured at the peak at a −90 mV holding current (AMPA). C and D, mean values of the AMPA/NMDA ratios in the developing and young adult stage, respectively. E, histogram showing the distribution of the AMPA/NMDA ratios in young (P14) mice. The distribution is skewed towards significantly lower AMPA/NMDA ratios in the Fmr1 KO2 mice.

Figure 3

Figure 3. AMPA/NMDA protein levels are reduced at P14 in Fmr1 KO2 mice

A, Western blot showing NR1, GluR-A and GluR-B levels at P14 in the homogenate and synaptoneurosome fractions. Shown are 2 pooled fractions from 2 mice each, for each genotype. For quantification of synaptoneurosomal GluR-A/NR1 protein levels, another 2 mice of each genotype were added to the analysis (not shown). Ten micrograms were loaded for each homogenate fraction (lane 2 is an internal loading control in which ½ amount was loaded). In order to obtain similar signal intensities as for the homogenate fraction for reliable quantification, only 15 μl of 500 μl of the synaptoneurosomal fraction was loaded. B, quantification of GluR-A/NR1 and GluR-B/NR1 protein amounts relative to the WT fraction (taken as 100%). C and D show AMPA receptor rectification. C, sample scaled traces showing AMPA-receptor-mediated currents in response to synaptic stimulation in the presence of an NMDA receptor blocker (

d

-APV, 30 μ

m

) in the recording medium and spermine in the patch pipette (black traces, WT; grey traces, Fmr1 KO2). Traces were obtained at holding currents of −40 to +40 mV, in 20 mV steps (_Y_-axis is current normalized to the value at −40 mV). D, summary data for 9 WT and 18 Fmr1 KO2 cells shows overlapping rectification curves of the AMPA-receptor-mediated synaptic responses.

Figure 1

Figure 1. Paired-pulse facilitation and intrinsic excitability are unaltered in Fmr1 KO2 mice

A, electrophysiological trace showing an overlay of four paired-pulse protocols scaled to the first stimulus, at 15–120 ms intervals. Scale bar: 1 fold is equivalent to no change. Values above stimulus artifacts denote initiation of second pulse at said interval (in ms) from first pulse. B, quantification of paired-pulse facilitation (PPF) at different interstimulus intervals. C displays sample traces showing similar intrinsic excitability in a WT control cell (upper panel) and an Fmr1 KO2 cell (lower panel).

Figure 4

Figure 4. Miniature currents are altered in Fmr1 KO2 mice

A, cumulative histogram showing the distribution of miniature AMPA currents in WT and Fmr1 KO2 mice. A significant shift is seen in the distribution. B, sample traces for A. A 3 s recording epoch is shown. C and D, same as for A and B, only for miniature NMDA currents. Here, too, a significant shift is seen in the cumulative histogram. Average rise (E), decay (half-width; F) and frequency (G) of AMPA miniature currents. There are no significant differences between genotypes. H, I and J, same as E–G, only for NMDA miniature events. Here, too, there are no significant differences between genotypes.

Figure 5

Figure 5. Long-term potentiation is increased in juvenile Fmr1 KO2 mice

A, induction of LTP at the hippocampal CA3–CA1 synapses using the LFS-LTP protocol in P14 mice. Filled squares represent WT and open squares Fmr1 KO2 test pathway values; filled and open circles represent control pathway. The duration of the induction protocol is omitted for clarity. Five minutes of baseline are shown, and potentiation following application of LFP started at time 0. Values are fold increase, after control-pathway subtraction (0 equals baseline). Insets show overlayed sample traces during baseline and 25 min after potentiation for WT and KO cells. B, as A, in 6- to 7-week-old mice. C, LTP induction using the theta-burst pairing (TBP) protocol. Input resistance changed < 10% for the duration of the recording. Five minutes of baseline are shown, and TBP was applied at time 0. Values are fold increase (1 equals baseline because there is no control-pathway subtraction). Insets 1 and 2 are sample traces illustrating the synaptic responses before and after LTP induction, and during potentiation. Inset 1, overlayed sample traces before and 30 min after potentiation showing the stimulus artifact, response of the cell to stimulation, and then response of the cell membrane voltage to a long depolarizing pulse for measuring input resistance. Inset 2, sample trace during the application of the TBP protocol. One train of a total of 30 is shown; stimulus artifacts can be seen 5 ms before a current injection evokes a postsynaptic spike. Five such pairings were repeated in each train, 10 times at theta frequency (5 Hz), and this was repeated for 3 times at 10 s intervals (Frick et al. 2004). D, LFS-LTP in C57BL/6 WT mice with and without the presence of an mGluR5 inhibitor, MPEP. Squares are test pathway, circles control pathway. Filled symbols are without, open symbols with MPEP.

Figure 6

Figure 6. Short- and long-term depression protocols in field and whole-cell recordings

Test (A) and control pathway response (B) to the LFS-depression protocol performed in field recordings according to Huber et al. (2002). Shown is a 10 min baseline, followed by a 15 min induction period (time points 0–15, indicated by black bar). The responses of both pathways to stimulation were monitored for another hour. C, control-subtracted values show identical response to LFS-depression protocol for both genotypes, and a return to baseline values about 30–35 min after induction (indicated by black bar), without overshooting. D, LFS-depression protocol in C57BL/6 mice without mGluR inhibitors (▪), with control pathway shown (□), and in the presence of

d

-APV (•), showing similar transient depression (lasting about 30 min post-LFS-depression protocol), and complete blockage by

d

-APV. E, sample trace showing the field EPSP (fEPSP) response of the test pathway followed by the response of the control pathway. The control pathway was recorded in stratum oriens and the detected fEPSP is therefore positive. For both pathways, the stimulus artifact can be seen, followed closely by the presynaptic fibre volley (easier to see for the test pathway owing to the direction of the stimulus artifact), and then the field response. F, depression protocol in patch-clamp whole-cell mode according to Terashima et al. (2008), showing no differences between WT (▪) and Fmr1 KO2 cells (□). Only STD was detected. G, depression protocol in patch-clamp whole-cell mode according to Zhang et al. (2005), showing no differences between genotypes. H, input–output curve and sample traces (I; from a WT slice). Fibre volley is easier to distinguish in I because of extended time scale.

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