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. 2003 Jul 23;552(Pt 1):35–45. doi: 10.1113/jphysiol.2003.045575

Impaired Regulation of Synaptic Strength in Hippocampal Neurons from GluR1-Deficient Mice

Bertalan K Andrásfalvy *, Mark A Smith *, Thilo Borchardt †, Rolf Sprengel †, Jeffrey C Magee *
PMCID: PMC2343312  PMID: 12878757

Abstract

Neurons of the central nervous system (CNS) exhibit a variety of forms of synaptic plasticity, including associative long-term potentiation and depression (LTP/D), homeostatic activity-dependent scaling and distance-dependent scaling. Regulation of synaptic neurotransmitter receptors is currently thought to be a common mechanism amongst many of these forms of plasticity. In fact, glutamate receptor 1 (GluR1 or GluRA) subunits containing L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors have been shown to be required for several forms of hippocampal LTP and a particular hippocampal-dependent learning task. Because of this importance in associative plasticity, we sought to examine the role of these receptors in other forms of synaptic plasticity in the hippocampus. To do so, we recorded from the apical dendrites of hippocampal CA1 pyramidal neurons in mice lacking the GluR1 subunit (GluR1 −/−). Here we report data from outside-out patches that indicate GluR1-containing receptors are essential to the extrasynaptic population of AMPA receptors, as this pool was nearly empty in the GluR1 −/− mice. Additionally, these receptors appear to be a significant component of the synaptic glutamate receptor pool because the amplitude of spontaneous synaptic currents recorded at the site of input and synaptic AMPA receptor currents evoked by focal glutamate uncaging were both substantially reduced in these mice. Interestingly, the impact on synaptic weight was greatest at distant synapses such that the normal distance-dependent synaptic scaling used by these cells to counter dendritic attenuation was lacking in GluR1 −/− mice. Together the data suggest that the highly regulated movement of GluR1-containing AMPA receptors between extrasynaptic and synaptic receptor pools is critically involved in establishing two functionally diverse forms of synaptic plasticity: LTP and distance-dependent scaling.

Virtually all CNS neurons involved in fast glutamatergic synaptic transmission express AMPA receptors and the great diversity of biophysical properties provided by these receptors makes them highly suitable for meeting many distinct physiological requirements (Wenthold et al. 1996; Geiger et al. 1997; Trussel, 1999). This variety of functional capabilities stems from the fact that most native AMPA receptors are composed of at least two different subunits (out of four GluR1-4 gene products) that form what is likely to be a tetrameric receptor-channel complex (reviewed in Dingledine et al. 1999). In addition, alternative splicing and RNA editing of receptor subunits adds to the diversity of receptor-channel compositions. Accordingly, specific expression patterns exist throughout the CNS with groups of neurons preferentially expressing one or two of the AMPA receptor subunits. For example, a prevalence of GluR4 has been found at fast auditory synapses (Trussel, 1999) while primarily GluR1-expressing neurons have been identified in cortex, striatum and spinal cord (Petralia & Wenthold, 1992; Furuyama et al. 1993; Martin et al. 1993; Tachibana et al. 1994). Hippocampal CA1 pyramidal neurons produce multiple AMPA receptor complexes that are composed of mainly GluR1- or GluR2-containing AMPA receptors (Wenthold et al. 1996).

GluR1-containing AMPA receptors are thought to be intimately involved in the regulation of synaptic strength in many neurons including CA1 neurons. Their ability to increase synaptic strength through either an activity-dependent delivery of new receptors (Hayashi et al. 2000; Shi et al. 2001; Passafaro et al. 2001; Lu et al. 2001; Piccini & Malinow, 2002) or an increase in single-channel conductance (Benke et al. 1998; Derkach et al. 1999) has been well documented in several associative forms of synaptic plasticity. It is also possible that a regulated synaptic delivery of AMPA receptors may be involved in modulations of synaptic strength occurring over longer timescales, such as homeostatic activity- and distance-dependent synaptic scaling (Inasek & Redman, 1973; Korn et al. 1993; Turrigiano et al. 1998; van Rossum et al. 2000; Turrigiano & Nelson, 2000; Andrásfalvy & Magee, 2001; Burrone et al. 2002; Smith et al. 2003). These separate forms of plasticity adjust overall synaptic strength to maintain a particular degree of synaptic influence over ongoing neuronal activity for any given level of postsynaptic excitability or synapse location (Turrigiano & Nelson, 2000; Magee, 2000). The role of GluR1-containing AMPA receptors in such longer timescale modulations of synaptic strength has, however, not been previously investigated.

In light of this, we have examined the impact of GluR1 deletion on the dendritic pools of AMPA receptors using a wide variety of recording techniques in hippocampal CA1 pyramidal neurons. Data presented here suggest that: (1) the extrasynaptic pool of AMPA receptors is almost exclusively composed of GluR1-containing receptors; (2) the synaptic pool of AMPA receptors also contains a large contingent of GluR1-containing receptors; and (3) a location-dependent insertion of GluR1-containing AMPA receptors produces distance-dependent scaling of Schaffer collateral synaptic strength in CA1 pyramidal neurons. Together these data further demonstrate the fundamental role that GluR1-containing AMPA receptors play in regulating synaptic strength in these neurons.

METHODS

Hippocampal slices and synaptic currents

According to methods approved by the Louisiana State University Health Science Center Institutional Animal Care and Use Committee, brains were rapidly removed after decapitation and placed in cold oxygenated low-Ca2+/high-Mg2+slicing solution. Hippocampal slices (350 μm) were prepared from 42-54-day-old GluR1 −/− mice and wild-type littermates (WT, C57BL6) using previously described standard procedures (Chen et al. 2001). All experiments in this study were performed blind as to genotype, using F6 and F7 generation littermates obtained from intercrosses of heterozygous animals that were derived from consecutive backcrosses into C57BL6 genetic background. Unless otherwise specified, the proximal recording location corresponds to a position where dendritic spine density had become substantial (50-75 μm from soma), and the distal location was a region approximately 30 μm from the termination of the stratum radiatum (180-220 μm from soma, Fig. 3A and B). Experiments were conducted using an upright Zeiss Axioskop microscope fitted with differential interference contrast (DIC) optics using infrared illumination. Patch pipettes (5-8 MΩ) were pulled from borosilicate glass and filled with an internal solution containing (mM): 120 caesium gluconate, 20 CsCl2, 0.5 EGTA, 4 NaCl, 0.3 CaCl2, 4 Mg2ATP, 0.3 Tris2GTP, 14 phosphocreatine and 10 Hepes (pH 7.2). The normal external solution contained (mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 25 dextrose, bubbled with 95 % O2-5 % CO2 at ≈33 °C (pH 7.4). All neurons had resting potentials between −60 and −75 mV. Series resistances from dendritic whole-cell recordings were between 10 and 30 MΩ. Unitary synaptic events were evoked by pressure ejection of a hyperosmotic external solution (with the addition of 300 mM sucrose), containing tetrodotoxin (TTX, 0.5 μM) and Hepes (10 mM) replacing NaHCO3(≈700 mosm l−1). AMPA currents were isolated by the presence of external D-aminophosphonovalerate (APV; 50 μM) and (+)-bicuculline (10 μM). Currents were recorded at −70 mV using an Axopatch 200B amplifier, filtered at 5 kHz and digitized at 50 kHz.

Figure 3. Distance-dependent scaling of synaptic current amplitude is attenuated in GluR1 −/− mice.

Figure 3

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Schematic diagrams of (A) proximal and (B) distal dendritic voltage-clamp recording configurations. C-F, representative recordings of hypertonically-evoked synaptic activity from proximal and distal dendrites in wild-type and in GluR1 −/− mice. G, averages of 100-165 individual mEPSCs from each of the recordings shown above. p and d represent proximal and distal recordings, respectively. H, grouped data of mean mEPSC amplitude for all cells. Numbers of cells are shown above each bar. I, mEPSC rise-time constants in GluR1 −/− mice are slightly faster than in WT mice, and this increase is independent of synaptic location (WT: 175 ± 9 μs, n = 11; KO: 134 ± 7 μs, n = 14, * P < 0.001). J, no differences are detected in mEPSC decay-time constants (WT: 4.3 ± 0.2 ms, n = 11; KO: 4.7 ± 0.4 ms, n = 14).

Miniature EPSCs crossing an approximate 4 pA threshold level were selected for further examination using a template-fitting algorithm written in Igor Pro (Magee & Cook, 2000; Smith et al. 2003). Events were fitted with a sum of two exponential functions to obtain peak amplitude, rise and decay-time constants. Events that had rise-time constants greater than 400 μs were eliminated from analysis since these events were unlikely to be from local synapses (Magee & Cook, 2000). Amplitude histograms were constructed from between 50 and 200 (typically 100-150) unitary events and were fitted with either dual or triple Gaussian functions. Goodness-of-fit was determined from the χ-squared values for each function. In pair-pulse facilitation experiments, electric stimulation was applied with a tungsten bipolar electrode (A-M systems Inc., Carlsborg, WA, USA) located ≈20 μm from the dendrite, at first proximal, then at a distal position from soma.

Two-photon glutamate uncaging

For MNI-glutamate (MNI-glu) uncaging experiments, hippocampal CA1 pyramidal neurons were visualized using an upright Olympus BX50WI microscope (Olympus America, Melville, NY, USA) fitted with a × 100, 1.0 NA water-immersion objective, as previously described (Smith et al. 2003). A mode-locked femtosecond-pulse Ti-sapphire laser (Coherent Inc., Auburn, CA, USA) was scanned with a modified confocal scan head (FluoView, Olympus America) and gated with a mechanical shutter at 4 ms (Uniblitz, Rochester, NY, USA). Hippocampal slices were incubated in the standard external solution in the presence of TTX (0.5 μM), APV (50 μM) and ascorbate (2 mM). Whole-cell dendritic recordings were made using patch pipettes containing a Ca2+-free standard caesium gluconate solution in the presence of ascorbate (2 mM) and bis-fura (150 μM; Molecular Probes Inc., Eugene, OR, USA) replacing EGTA. Dendrites were voltage-clamped at −70 mV using an Axopatch 1D amplifier (Axon Instruments) and had access resistances of 15-30 MΩ. In addition, a broken pipette containing the standard external solution in the presence of TTX (0.5 μM), APV (50 μM), ascorbate (2 mM), cyclothiazide (100 μM) and caged MNI-glu (12 mM) was positioned above the slice at the site of the recording electrode. All spines examined were between 20-30 μm below the surface of the slice.

The point spread function of focal volume for two-photon excitation, estimated using 0.1 μm fluorescent beads, was 0.34 μm laterally and 1.4 μm axially (full-width half-maximum; FWHM). The FWHM of glutamate current amplitude distributions obtained by point uncaging of MNI-glu (720 nm, 7 mW) at different locations (≈0.16 μm lateral and 0.5 μm axial steps) on well-isolated spines was 0.6 μm laterally and 2.0 μm axially. Isolated spines were identified on the basis that no other spine was within 1 μm of the examined spine in the same lateral plane and no other spine was directly below or above this perimeter (Smith et al. 2003). For spines showing a head diameter greater than 0.4 μm, head volume was estimated directly from the spine head diameter determined from fluorescence point spread functions (FWHM) and substituted into the equation for a sphere, Volume = (4πr3)/3, where r is the spine head radius. For spines with a smaller diameter, volume was estimated by referencing total fluorescence of the spine head acquired from three-dimensional reconstructions to the total fluorescence of a large (> 0.4 μm) spine head whose volume could be estimated as above (Smith et al. 2003).

Rapid glutamate application

Outside-out patches were pulled from dendritic locations by patch pipettes filled with an internal solution containing (mM): 140 KMeSO4, 1 BAPTA, 10 Hepes, 4 NaCl, 0.28 CaCl2, 4.0 Mg2ATP, 0.3 Tris2GTP, 14 phosphocreatine and 0.05 spermine (pH 7.25). Fast application of agonist was performed and the data acquisition with analysis was executed as previously described (Andrásfalvy & Magee, 2001). Initially, 10 ms pulses of 1 mM glutamate were applied with 10 μM glycine to evoke the AMPA and NMDA receptor-mediated currents. Then the solution was switched to a 1 mM MgCl2-containing solution, in the absence of glycine, to isolate the AMPA receptor current. AMPA receptor currents were subtracted from the whole glutamate current to measure the NMDA receptor component. Kinetic features of AMPA receptor-mediated glutamate currents were acquired with 1 and 100 ms, 1 mM glutamate applications. Currents were recorded at –80 mV using an Axopatch 200B amplifier, filtered at 2 kHz and digitized at 20 kHz.

RESULTS

AMPA current in dendritic outside-out patches

We began by assessing the impact of GluR1 subunit deletion on the dendritic AMPA receptor population. To do so we compared currents produced by the rapid application of glutamate to outside-out patches excised from different dendritic regions of hippocampal CA1 pyramidal neurons from both wild-type (WT) and GluR1 −/− (KO) mice (Fig. 1A-D). The AMPA component of patches excised from a location where there are essentially no excitatory synapses (≈20 μm from the soma) was decreased by approximately 97 % in GluR1 −/− mice (WT: 541 ± 108 pA, n = 9; KO: 11 ± 2 pA, n = 9; P < 0.001, Fig. 1E). A similar magnitude of reduction was also detected at a more distal region (170-200 μm) where spine and synaptic density is high (WT: 874 ± 136 pA, n = 8; KO: 25 ± 8 pA, n = 12; P < 0.001, Fig. 1E).

Figure 1. The extrasynaptic pool of AMPA and NMDA receptors is severely altered in GluR1 −/− mice.

Figure 1

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Representative AMPA and NMDA receptor current traces, evoked by rapid glutamate applications to dendritic outside-out patches, are shown for proximal (A and C) and distal patches (B and D) from wild-type (A and B) and GluR1 −/− mice (C and D). Currents are the averages of 3-5 individual traces. Mean AMPA (E) and NMDA (F) receptor current amplitudes are shown for all groups. Representative non-stationary fluctuation analysis (NSFA) of distal patches from WT (G) and KO (H) mice. Note that patches from KO mice have a dramatic reduction in receptor number (N), and maximum open-probability (Po,max) (I), whilst the single-channel conductance (γ) is the same in both groups (J). K, current-voltage relationships of AMPA receptor currents are similar between WT (squares) and KO (circles) mice. WT: Erev = 5.0 ± 0.5 mV, n = 11; KO: Erev = 2.0 ± 1.0 mV, n = 4.

That AMPA receptor currents from both spiny (synaptic) and non-spiny (extrasynaptic) regions were reduced by comparable magnitudes suggests either that the receptors in all outside-out patches were from extrasynaptic regions or that synaptic and extrasynaptic currents were reduced by equally large amounts. Previous data (Zamanillo et al. 1999) as well as data presented below strongly favour the former interpretation. Nevertheless, the hypothesis that outside-out patches contain only extrasynaptic receptors is in contrast with our previous claim that relied heavily on the now unlikely assumption that cycling between extrasynaptic and synaptic NMDAR pools is slow (see Andrásfalvy & Magee, 2001; Tovar & Westbrook, 2002).

Interestingly, the NMDA receptor component from GluR1 −/− mice appeared to be somewhat reduced in both dendritic regions by approximately 50 %, implying a linkage between AMPA and NMDA receptor numbers in the extrasynaptic receptor pool (Lisman & Zhabotinsky, 2001; Fig. 1F). However, this result should be taken with some caution as the greatly reduced size of the AMPA currents in the KO mice may have produced some inaccuracies in the current-subtraction procedure adversely affecting the size of the NMDA component.

Channel subunit composition

Channel subunit composition has been shown to play a determining role in many of the basic properties of various agonist-gated ion channels, including AMPA receptors (Verdoorn et al. 1991; Mosbacher et al. 1994; Swanson et al. 1997). Therefore to examine this more closely we compared the AMPA receptor current kinetics, voltage dependence and single-channel properties in patches from WT and KO mice. From these patches we saw that current rise-time constants were not altered, while AMPA receptor deactivation-time constants and the fast component of desensitization were significantly faster in GluR1 −/− mice (Fig. 2). Also the fast component of desensitization was much more prominent in patches from KO mice (Fig. 2D, F and G). Furthermore, non-stationary fluctuation analyses indicated that the calculated maximum open-probability (Po,max) was heavily reduced in KO mice while single-channel conductance (γ) was identical from both locations in WT and KO mice (Fig. 1G and H). Finally, current-voltage relationships were very similar in both sets of mice except for a slight shift in reversal potential (WT: Erev = 5.0 ± 0.5 mV, n = 11; KO: Erev = 2.0 ± 1.0 mV, n = 4; P < 0.001 Fig. 1K). All of the above AMPA receptor properties were independent of patch location.

Figure 2. Some kinetic properties of AMPA currents are altered in GluR1 −/− mice.

Figure 2

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A, rise times of outside-out patch currents are not altered (WT: 457 ± 34 μs, n = 16; KO: 568 ± 50 μs, n = 8; P > 0.05), whilst, as shown in B, the deactivation-time constants (WT: 3.2 ± 0.2 ms, n = 8; KO: 2.2 ± 0.2 ms, n = 12, * P < 0.001) and, as shown in D, the fast component of desensitization (WT: 8.1 ± 0.7 ms, n = 8; KO: 5.6 ± 0.4 ms, n = 12, * P < 0.05) are significantly faster in GluR1 −/− mice. G, the fast component of desensitization is also much more prominent KO mice (WT desensitization ratio 22 ± 2 %; KO desensitization ratio 82 ± 20 %, * P < 0.001). Representative AMPA receptor currents, evoked by 1 (C) and 100 ms (F) glutamate pulses (1 mM), are shown for WT and KO dendritic patches.

AMPA receptors that lack the GluR2 subunit rectify and are calcium permeable (except for GluR2long), whilst homomeric GluR2 receptors have an extremely small single-channel conductance. These properties contrast with the linear current-voltage relationships and the relatively large γ values obtained in both sets of mice. These data therefore suggest that AMPA receptors in adult wild-type mice are composed of heteromeric GluR1/2 and/or GluR2/3 subunits. That the extrasynaptic pool was virtually absent in the GluR1 −/− mice suggests that, under control conditions, the vast majority of the receptors in the extrasynaptic pool of wild-type mice are GluR1/2 heteromers with small amounts of GluR2/3 heteromers also present.

Synaptic currents and distance-dependent scaling

As mentioned above, the cycling of AMPA receptors (Nishimune et al. 1998; Lüthi et al. 1999; Lüscher et al. 1999) between extrasynaptic and synaptic pools is thought to be a critical component in many forms of synaptic plasticity (Zamanillo et al. 1999; Lu et al. 2001; Piccini & Malinow, 2002; Andrásfalvy & Magee, 2002). The nearly complete loss of the extrasynaptic pool of AMPA receptors in the GluR1 −/− mice strongly suggests that this receptor cycling should be essentially absent in CA1 pyramidal neurons from these mice. We therefore tested the idea that cycling plays a fundamental role in establishing distance-dependent scaling by comparing the location dependence of synaptic currents in CA1 neurons from both WT and KO mice. Whole-cell recordings from different regions of the apical dendritic arbor were used to record miniature excitatory postsynaptic currents (mEPSCs) evoked by the localized application of a high-osmolarity external solution (see Methods and Fig. 3A and B; Magee & Cook, 2000; Smith et al. 2003). Consistent with data obtained from rats (Magee & Cook, 2000; Smith et al. 2003), distal mEPSC amplitudes in WT mice were approximately twofold larger than proximal events (proximal: 11.7 ± 0.9 pA, n = 5 vs. distal: 24.2 ± 2.3 pA, n = 6, Fig. 3C and E). GluR1 −/− mice, on the other hand, exhibited a greatly reduced distance-dependent scaling of synaptic amplitude (proximal: 8.2 ± 0.8 pA, n = 6 vs. distal: 10.1 ± 0.9 pA, n = 8, P > 0.05; Fig. 3D and F), as compared to wild-type mice (WT: 107 % increase vs.KO: 23 % increase). Also, mEPSC rise-time constants in GluR1 −/− mice were found to be slightly faster than in WT mice whilst no differences were detected in mEPSC decay-time constants (Fig. 3I and J). The mEPSC data show that GluR1-containing AMPA receptors are a significant component of the synaptic pool of AMPA receptors at Schaffer collateral (SC) synapses and furthermore that GluR1 receptors are required for distance-dependent scaling in CA1 pyramidal neurons. The loss of this compensatory modulation of synaptic strength causes a location-dependent reduction in basal synaptic strength in GluR1 −/− mice with proximal synapses experiencing much less attenuation than more distal synapses. Thus, as discussed below, it is possible that a location-dependent difference in the proportion of GluR1-containing AMPA receptors exists at SC synapses under normal conditions.

The reduction in mEPSC amplitude reported here for distal SC synapses contrasts with a previous report that observed no differences in synaptic weight between WT and GluR1 −/− mice (Zamanillo et al. 1999). The reason for this discrepancy is likely to be due to the differences in recording location (i.e. somatic vs.dendritic) between the studies. By recording from the soma and using a relatively rapid rise-time cutoff, most of the synaptic currents recorded by Zamanillo et al. (1999) would have come from proximal synapses and therefore not show much change in amplitude.

GluR1 and quantal size

To determine the mechanism by which GluR1-containing AMPA receptors modulate the basic synaptic weight of SC synapses we performed additional analyses of the synaptic currents. Further examination of the current data revealed that the distribution of mEPSC amplitudes was highly variable in all groups (CV > 50 %, Fig. 4A). Whilst no differences in the coefficients of variation were observed for either synapse location or mouse type, a large location dependence was observed for the parameter σ2/x (the variance divided by the mean) only in the WT mice (Fig. 4B). These data suggest that there is an increase in the quantal size at distal WT synapses that does not occur in GluR1 −/− mice. Furthermore, the large amount of positive skew (skewness: 1.2 ± 0.03) observed in the amplitude distributions suggests that they might be composed of multiple components (Fig. 4G). In fact the distributions from most neurons were well fitted by multiple Gaussian functions, with peak separation increasing with distance, again, in only the WT mice (see Methods, Fig. 4C-G). Finally, cumulative frequency distributions demonstrate that distal synaptic inputs from WT mice are uniformly shifted towards larger amplitudes, whilst proximal WT and both proximal and distal distributions from KO mice almost overlap at lesser amplitudes (Fig. 4H). Together these statistical data suggest that the amplitude of distal mEPSCs are increased in WT mice by a scaling-up of either postsynaptic AMPA receptor effectiveness or the amount of glutamate packaged into a synaptic vesicle. That this scaling is not found at distal synapses of GluR1 −/− mice points strongly towards an effect mediated though postsynaptic receptors and not presynaptic vesicular content, also the lack of any significant differences in pair-pulse facilitation ratios amongst the groups further supports this interpretation (Fig. 4I; Zamanillo et al. 1999). The evidence thus far presented points towards the idea that a location-dependent delivery of GluR1-containing AMPA receptors increases the quantal size of distal SC synapses, resulting in a distance-dependent scaling of synaptic weight.

Figure 4. Synaptic current distributions from GluR1 −/− mice show altered properties.

Figure 4

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A, plot of CV for all four groups of mEPSCs. Numbers shown on plot are mean 1/CV2, a measure that is indicative of quantal content. B, plot of σ2/x for the four groups of mEPSCs. This measure is indicative of quantal size and the plots indicate that the normal distance-dependent increase in this parameter is absent in GluR1 −/− mice. Increases in σ2/x from proximal locations to distal (distal/prox) are displayed on the plot. C-F, mEPSC amplitude distributions of proximal and distal synapses from wild-type (WT) and knockout mice (KO). Between 135 and 204 events were plotted using a bin size of 1 pA. Solid lines are fits of data by the sum of three Gaussians with individual mean peaks at WT-prox x1 = 6, x2 = 10, x3 = 20 pA; WT-distal x1 = 13, x2 = 25, x3 = 42 pA; KO-prox x1 = 4, x2 = 8, x3 = 15 pA; KO-distal x1 = 6, x2 = 12, x3 = 17 pA. G, plot of mean peak amplitudes from the multiple Gaussian fits for each group of distributions. Numbers on the plot are the ratio of WT distal to all other group peak amplitudes, suggesting again that distal synapses in GluR1 −/− mice lack an approximately 2.5-fold increase in quantal size. H, cumulative frequency distribution of all events from WT (blue) and GluR1 −/− (red) mice. Continuous lines are proximal and dashed lines are distal distributions. I, pair-pulse facilitation ratios (EPSC2nd/EPSC1st) are shown at four different interstimulus intervals, recorded and stimulated at proximal (filled circle) and distal (open circle) dendritic regions, from WT (blue) and KO (red) mice. Prox, proximal.

Synaptic AMPA receptor currents

To further examine the possible mechanisms of the loss of distance-dependent scaling in KO mice, the location dependence of synaptic AMPA receptor currents was determined by focally applying glutamate onto isolated dendritic spines using multi-photon uncaging of MNI-glutamate (Matsuzaki et al. 2001; Smith et al. 2003). Glutamate was released near well-isolated spines either on the dendritic trunk or radial oblique dendrites that were within 20 μm of the dendritic recording pipette (Fig. 5A-D). In wild-type mice, the mean AMPA receptor-mediated current obtained from proximal spines was 32 ± 4 pA (16 spines from seven cells; spine volume: 0.11 ± 0.01 μm3) compared with 69 ± 4 pA at distal spines (13 spines from six cells; spine volume: 0.10 ± 0.01 μm3) (Fig. 5F). Therefore, as previously observed in rats, distal spines from wild-type mice show a nearly twofold increase in AMPA receptor-mediated glutamate responsiveness when compared to proximal spines (Smith et al. 2003). However this large distance-dependent increase in AMPA receptor current was not observed in the spines of KO mice (P > 0.05), such that proximal amplitudes were 26 ± 3 pA (13 spines from six cells; spine volume: 0.10 ± 0.01 μm3) and distal amplitudes were 33 ± 2 pA (14 spines from six cells; spine volume: 0.11 ± 0.01 μm3; Fig. 5F). In all groups, the glutamate currents had similar rise and decay-time constants (see Fig. 5G and H).

Figure 5. The synaptic pool of AMPA receptors is reduced in a location-dependent manner in GluR1 −/− mice.

Figure 5

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A, image stack spanning the entire apical dendritic arborization of another CA1 pyramidal neuron. Ovals represent distal and proximal recording sites in stratum radiatum (S. Rad). Dashed lines demonstrate the proximal stratum pyramidale (S. Pyr.) and the distal stratum lacunosum-moleculare (S. Lac. Mol.) borders of stratum radiatum. B, location-dependent AMPA currents from WT and KO mice, averages of 2-3 traces. Traces are fitted by the sum of two exponentials, with blue lines for WT and red for KO mice. Note that the distance-dependent increase in AMPA current is missing in the recordings from the KO mice. C, multi-photon image stack of a distal dendritic region of a CA1 pyramidal neuron filled with bis-fura2. The dendritic recording electrode is shown on the left and the coloured arrows indicate the isolated spines that gave the correspondingly coloured MNI-glu currents shown to the right. D, examples of AMPA receptor currents evoked by focal uncaging of MNI-glutamate (MNI-glu) onto isolated spines located on the main dendritic trunk and a nearby oblique dendrite (branch to the right), as indicated by coloured arrows shown in C. E, mean AMPA current amplitudes for wild-type (blue bars) and GluR1 −/− (red bars) mice. F, the spine head volumes were the same in both groups of mice in both locations. G and H, in all groups of MNI-glu currents have similar time constants of rise (WT: 1.95 ± 0.1 ms, n = 29; KO: 2.0 ± 0.1 ms, n = 23) and decay (WT: 5.85 ± 0.4 ms, n = 29; KO: 6.1 ± 0.5 ms, n = 23).

Therefore, the distance-dependent increase in postsynaptic AMPA receptor current that is normally found in these neurons is absent in the GluR1 −/− mice, just as was observed above for the spontaneous synaptic currents. The observed reduction in the spine AMPA current in KO mice is comparable to that seen in mEPSC amplitude and is much less than that observed in the dendritic patches, thus lending credence to the idea that most of the AMPA receptors activated by glutamate uncaging are synaptic in origin while those in the patches are from extrasynaptic sources (Fig. 6A and B). These data strengthen the idea that distal Schaffer collateral synapses normally contain a higher density of AMPA receptors that increase the responsiveness of the synapse to quantal glutamate release and furthermore that this increase is dependent upon the presence of GluR1-containing AMPA receptors.

Figure 6. Comparison of alterations in the extrasynaptic and synaptic receptor pools.

Figure 6

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A, decrease in amplitude observed in GluR1 −/− mice for AMPA currents recorded in outside-out patches (patches), glutamate uncaging (MNI-glu) and mEPSCs. Note that mEPSC and MNI-glu currents are similarly reduced whilst patch currents are reduced to a much greater extent. p, proximal currents; d, distal currents. B, plot of the increase in distal current amplitude for mEPSCs and MNI-glu currents in both wild-type and GluR1 −/− mice. Notice the increases in mEPSCs amplitudes are mirrored by the increases in MNI-glu current.

DISCUSSION

Summary

We have characterized the properties of dendritic AMPA receptors, spontaneous synaptic currents and postsynaptic AMPA receptor responsiveness in hippocampal CA1 pyramidal neurons from both wild-type and GluR1 −/− mice. The main observations are: (1) AMPA receptor currents from outside-out patches pulled from the apical dendrites of GluR1 −/− mice are severely reduced in amplitude. (2) These currents from the KO mice also appear to decay faster and have a lower probability of opening than WT currents. (3) Spontaneous synaptic currents are also smaller in amplitude in the KO mice and the degree of this reduction is dependent upon the dendritic location of the synapse, with distal synapses showing the greatest reduction. (4) Statistical analyses of the synaptic currents indicate that the distal SC synapses of KO mice lack a normal increase in postsynaptic responsiveness, and focal application of glutamate onto postsynaptic spines confirms this scenario. We interpret these data to indicate that the extrasynaptic pool of AMPA receptors is almost entirely composed of GluR1-containing AMPA receptors (probably GluR1/2 heteromers) and that distance-dependent scaling of SC synaptic weight is the result of an increased delivery of these receptors to distal synapses. Furthermore, this regulated delivery probably involves receptor movement between the synaptic and extrasynaptic pools of AMPA receptors.

Increased density of GluR1 at distal synapses

The similarities in the ratios of distal to proximal mEPSCs and postsynaptic AMPA receptor currents in both WT and KO mice (Fig. 6B), as well as the lack of any significant differences in pair-pulse facilitation ratios (Fig. 4I; Zamanillo et al. 1999), all minimize the likelihood that an alteration in presynaptic vesicular release is responsible for the loss of synaptic scaling in GluR1 −/− mice. Instead, the spine-level differences in AMPA receptor currents, revealed by multi-photon glutamate uncaging, suggest that distal synapses of WT mice have either greater numbers or more effective AMPA receptors. We suspect that the number of AMPA receptors is elevated at distant synapses because we have observed such an increase in rat hippocampal CA1 pyramidal neurons (Smith et al. 2003). Accordingly the impaired glutamate responsiveness observed in GluR1 −/− mice seems to indicate that a deficiency in the location-dependent insertion of AMPA receptors causes distance-dependent scaling of basal synaptic weight to be impaired in these mice. Having said this, it is possible that the differences in Po,max observed in the outside-out patch currents could also produce location-dependent synaptic currents if there were differences in the proportion of the higher Po,max GluR1-containing heteromers present at the synapses. In this case if distal synapses contain relatively more GluR1-containing AMPA receptors compared to more proximal synapses, the higher Po,max of those AMPA receptors could add additional weight to the distal synapses. Given the data from rat CA1 cells, however, we find it more likely that the main mechanism of synaptic scaling is an increased delivery of GluR1-containing AMPA receptors that in turn might also have a higher Po,max.

There are, however, alternative interpretations revolving around the idea that the reduced GluR1 activity in the KO mice somehow inhibits distance-dependent scaling without any relation to the direct insertion of GluR1-containing AMPA receptors (AMPARs) into the synapse. One of these interpretations would be that the reduced depolarization of distal synapses in KO mice would preclude the development of distance-dependent scaling. This will of course depend on the exact signalling mechanisms responsible for this form of plasticity, which are at present unknown. However, we feel that it is in fact the intrinsically smaller impact of distal synapses, due to dendritic filtering, that produces the scaling to begin with and that, as this impact will be even smaller in the KO mice, scaling should still occur in these mice as long as the delivery mechanism still exists. It is our proposition that this delivery mechanism does in fact not exist without GluR1-containing AMPARs. Another related idea is that the GluR1-containing AMPARs are, in some way, required for the delivery of other (GluR2/3) AMPARs into the synapse. It is difficult for us to exclude this interpretation except that it is perhaps less straightforward than the idea of a regulated insertion of GluR1-containing (GluR1/2) AMPARs into the synapse.

AMPA receptor cycling and homeostatic synaptic plasticity

Previous studies have demonstrated subunit-specific AMPA receptor transportation routes where GluR2/3 receptors are delivered by direct insertion and continuous recycling within the synapse. Modulations of this cycling are thought to set basal synaptic strength and underlie some forms of LTD (Shi et al. 2001; Kim et al. 2001). GluR1/2 receptors, on the other hand, are thought to initially insert into the extrasynaptic membrane and then move laterally into the postsynaptic density depending on the activity pattern of the synapse (Lu et al. 2001; Piccini & Malinow, 2002; Andrásfalvy & Magee, 2002). This scheme is supported by observations that GluR1 −/− mice lack both a significant extrasynaptic AMPA receptor pool as well as certain forms of associative LTP (Zamanillo et al. 1999; Mack et al. 2001; but see Hoffman et al. 2002). That these mice also lack distance-dependent scaling suggests that longer timescale modulations of synaptic strength also use the same GluR1-containing AMPA receptor delivery system to regulate basal Schaffer collateral strength.

Although both of these diverse forms of plasticity appear to use the same receptor delivery system, the induction rules governing the location-dependent addition of GluR1/2 receptors would not be the same as those seen in most forms of associative synaptic plasticity (where brief positive associations produce increased strength). Instead, there must be some variation of the standard activity-dependent induction rules that is perhaps based on the differences in timescale (where positive associations over many hours produce decreased strength; L. Abbott, personal communication). Although the exact control mechanisms are still murky, it is clear that the highly regulated movement of GluR1-containing AMPA receptors is fundamental to the modulation of synaptic strength in CA1 pyramidal neurons and, furthermore, that this modulation is critical for proper hippocampal functioning (Reisel et al. 2002).

Acknowledgments

This work was supported by National Institutes of Health Grant NS 35865 and NS 39458. We thank Graham Ellis-Davies, Drexel University, Philadelphia, USA for MNI-glu and Larry Abbott, Brandeis University, Waltham, USA for helpful discussions.

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