The delta2 'ionotropic' glutamate receptor functions as a non-ionotropic receptor to control cerebellar synaptic plasticity

Abstract

The delta2 glutamate receptor (GluRdelta2) belongs to the ionotropic glutamate receptor (iGluR) family and plays a crucial role in the induction of cerebellar long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Nevertheless, the mechanisms by which GluRdelta2 regulates cerebellar LTD have remained elusive. Because a mutation occurring in lurcher mice causes continuous GluRdelta2 channel activity that can be abolished by 1-naphtylacetylspermine (NASP), a channel blocker for Ca(2+)-permeable iGluRs, GluRdelta2 is thought to function as an ion channel. Here, we introduced a mutant GluRdelta2 transgene, in which the putative channel pore was disrupted, into GluRdelta2-null Purkinje cells using a virus vector. Surprisingly and similar to the effect of the wild-type GluRdelta2 transgene, the mutant GluRdelta2 completely rescued the abrogated LTD in GluRdelta2-null mice. Furthermore, NASP did not block LTD induction in wild-type cerebellar slices. These results indicate that GluRdelta2, a member of the iGluR family, does not serve as a channel in the regulation of LTD induction.

Figure 1. Mutations known to disrupt the channel pores of AMPA/kainate receptors effectively suppressed the current flow through GluRδ2^Lc channels in HEK 293 cells

A, presumed membrane topology of GluRδ2. Glutamine (Q) at the Q/R site and a negatively charged glutamate (E) are shown, which are presumed to be exposed to the channel pore and to determine its sensitivity to spermine analogues. B, alignment of the amino acid sequences of the channel pore regions of representative iGluRs. GluRδ2 has a Q at the Q/R site (position 0). The conserved glycine (G) residue at position +2 from the Q/R site contributes to the narrow constriction of the iGluR channel. Letters are shaded according to the similarity of the amino acids at each position. C–I, representative current–voltage relationships recorded from naïve HEK 293 cells (Naïve; panel C), or HEK 293 cells expressing an empty vector (Vector; panel D), GluRδ2^Lc (panel E), GluRδ2^Lc in which alanine (G/A, panel F), phenylalanine (G/F, panel G) or tryptophan (G/W, panel H) were substituted for glycine (G) at position +2, or GluRδ2^Lc in which arginine was substituted for valine (V) at position −1 (V/R, panel I). The thin traces shown in panels F–I indicate the current–voltage relationship of cells expressing GluRδ2^Lc (taken from panel E); the thin dashed traces indicate the current–voltage relationship of cells expressing empty vector (taken from panel D). Insets of panel E–H: side-chain structures of the replaced amino acids. J, bar graph showing the mean current density at −80 mV. The number of experiments is given in parentheses. **P < 0.01.

Figure 2. Virally mediated expression of GluRδ2 mutants with disrupted channel pores rescued abrogated cerebellar LTD in GluRδ2-null mice

A, confocal images of the virus-infected GluRδ2-null cerebellum. Infected Purkinje cells were identified using nYFP fluorescence (top). Transduced GluRδ2^wt-V/R was visualized using anti-GluRδ2 antibodies and Alexa 546-conjugated secondary antibodies (middle) and merged with the nYFP signals (bottom). Each right panel shows a magnified view. B–E, PF-EPSC amplitudes following LTD-inducing conjunctive stimulation (CJ-stim) recorded from GluRδ2-null Purkinje cells expressing empty vector (+ Vector; B), GluRδ2^wt (+ GluRδ2^wt; C), or GluRδ2^wt-V/R (+ GluRδ2^wt-V/R; D). Averaged data are shown in E. Inset traces: PF–EPSCs just before (1) and 30 min after (2) CJ-stim and their superimposition (1 + 2).

Figure 3. Suppression of LTD in GluRδ2-null Purkinje cells expressing GluRδ2^wt-V/R by a peptide mimicking the C-terminus of GluRδ2

A, schematic diagram of the experimental set-up. GluRδ2-null Purkinje cells transduced with GluRδ2^wt-V/R were perfused with pep-GluRδ2CT7, a peptide that matched the C-terminal 7 amino acids of GluRδ2, and pep-GluRδ2SCR, a peptide in which the sequence of pep-GluRδ2CT7 was scrambled, via a recording (Rec.) patch pipette. B and C, representative LTD data recorded from GluRδ2-null Purkinje cell expressing GluRδ2^wt-V/R perfused with pep-GluRδ2CT7 (500 μm; B) or pep-GluRδ2SCR (500 μm; C). Inset traces: PF-EPSCs recorded at times 1 and 2. D, averaged LTD data.

Figure 4. Channel blockers of GluRδ2 did not hamper LTD induction in wild-type cerebellum

A, current–voltage curves recorded from HEK 293 cells expressing GluRδ2^Lc before (−) and after (+) the application of NASP (10 μm). The thin dashed trace indicates the representative current–voltage relationship of cells expressing an empty vector (taken from Fig. 1D) for comparison. B, bar graph showing the mean current density at −80 mV; *P < 0.05. C, confocal image of Bergmann glia visualized by the inclusion of Alexa 488-conjugated dextran in a patch pipette. D, AMPA-induced current responses from Bergmann glia before (thick traces; holding potential at +40 mV and −60 mV) and after (thin trace; holding potential at −60 mV) the application of 100 μm of NASP. AMPA currents were evoked by puffing (RS)-AMPA (10 mm) to the soma of Bergmann glia in the presence of cyclothiazide (50 μm). E and F, representative (E) and summarized (F) LTD data recorded from wild-type cerebellar slices in the presence of NASP (100 μm). Inset traces: PF-EPSCs recorded at times 1 and 2.