Intracellular Ca²⁺ and not the extracellular matrix determines surface dynamics of AMPA-type glutamate receptors on aspiny neurons

Abstract

The perisynaptic extracellular matrix (ECM) contributes to the control of the lateral mobility of AMPA-type glutamate receptors (AMPARs) at spine synapses of principal hippocampal neurons. Here, we have studied the effect of the ECM on the lateral mobility of AMPARs at shaft synapses of aspiny interneurons. Single particle tracking experiments revealed that the removal of the hyaluronan-based ECM with hyaluronidase does not affect lateral receptor mobility on the timescale of seconds. Similarly, cross-linking with specific antibodies against the extracellular domain of the GluA1 receptor subunit, which affects lateral receptor mobility on spiny neurons, does not influence receptor mobility on aspiny neurons. AMPARs on aspiny interneurons are characterized by strong inward rectification indicating a significant fraction of Ca(2+)-permeable receptors. Therefore, we tested whether Ca(2+) controls AMPAR mobility in these neurons. Application of the membrane-permeable Ca(2+) chelator BAPTA-AM significantly increased the lateral mobility of GluA1-containing synaptic and extrasynaptic receptors. These data indicate that the perisynaptic ECM affects the lateral mobility differently on spiny and aspiny neurons. Although ECM structures on interneurons appear much more prominent, their influence on AMPAR mobility seems to be negligible at short timescales.

Keywords: extracellular matrix; glutamate receptor; interneurons; single particle tracking.

Figure 1.

The lateral mobility of GluA1 and GluA2 around aspiny synapses. (a,b) Dual labelling of Homer 1c (green) and GluA1 (magenta) in a spiny (a) and an aspiny (b) neuron. Boxed regions are magnified on the right. Scale bars apply to (a) and (b). (c) Quantification of synaptic live-staining for GluA1 (upper panel) and GluA2 (lower panel) AMPAR subunits in spiny and aspiny neurons. Data are shown as mean ± s.e.m., p < 0.01, t-test. (d,e) Distribution of instantaneous diffusion coefficients (D_inst) for synaptic (syn) and all trajectories (all) of endogenous GluA1 (d) and GluA2 (e) subunits in aspiny neurons obtained in SPT experiments ((d) synaptic: n = 143, all: n = 928 trajectories, eight cells, (e) synaptic: n = 882, all: n = 10 937 trajectories, two cells). (f,g) Box-plots show D_inst for the mobile fraction (D > 0.001 µm² s⁻¹) of GluA1 (f) and GluA2 (g) in aspiny neurons under control conditions and after exposure to hyaluronidase (HYase). Data are shown as median/interquartile range, p < 0.005; Mann–Whitney test.

Figure 2.

ECM degradation has no effect on dynamic and subunit composition of AMPARs and short-term plasticity in aspiny neurons, whereas change of intracellular free calcium alters AMPAR dynamics. (a) Rectification index (RI) of AMPAR populations in aspiny synapses measured in control, after ECM digestion by hyaluronidase (HYase), after cross-linking of GluA1 (X-link) and in combination of ECM-digestion and cross-linking (Hyase + X-link, p > 0.05, t-test). (b) Plot of the recovery from desensitization of AMPAR eEPSC induced by iontophoretic Glu application in aspiny neurons as a function of the interstimulus interval (ISI) under indicated conditions. Number of cells in each group are: control = 21, HYase = 11, X-link = 11, HYase + X-link = 9 (F = 1.84, p > 0.05, two-way ANOVA). Inset demonstrates no difference in the decay of eEPSCs under indicated conditions. Data are shown as mean ± s.e.m. (c) Mean ± s.e.m. for amplitude of eEPSCs under indicated conditions (p > 0.05 for all comparisons, Kruskal–Wallis test). (d) Plot of the recovery from desensitization of synaptic (syn) and extrasynaptic (extra) eEPSCs in aspiny neurons in the presence of 500 µM kynurenic acid (Kyn, n = 5, F = 14.22; p < 0.01, two-way ANOVA). Inset demonstrates absence of difference in the decay between synaptic and extrasynaptic eEPSCs. (e,f) FRAP experiment. (f) Normalized fluorescence recovery curve of GluA1::SEP bleached in aspiny synapses under control conditions and after treatment with HYase. (f) Mean ± s.e.m. of averaged normalized recovery in aspiny neurons at 300 s after photobleaching for synaptic (syn) and extrasynaptic locations (dend) without and after HYase treatment for neurons expressing either GluA1::phluorin or GluA2::phluorin as indicated. Number of bleached spots indicated in bars. Comparing synapses or dendrites before and after HYase treatment indicated significant differences as indicated between synaptic and dendritic compartments, t-test. (g,h) Box-plot of the median/interquartile range of instantaneous diffusion coefficients (D_inst) for synaptic (syn) and total (all) GluA1 fractions in aspiny (g) and spiny (h) neurons incubated without or with BAPTA-AM or philantotoxin433 (PhTx433). BAPTA-AM and PhTx433 increased the mobility (D_inst) of synaptic and total fraction of GluA1 in aspiny but not in spiny neurons (p < 0.01 for synaptic and p < 0.005 for all trajectories, Mann–Whitney test).