Modification of NMDA receptor channels and synaptic transmission by targeted disruption of the NR2C gene

Abstract

A novel strain of mutant mouse has been generated with a deletion of the gene encoding the NR2C subunit of the NMDA receptor, which is primarily expressed in cerebellar granule cells. Patch-clamp recordings from granule cells in thin cerebellar slices were used to assess the consequences of the gene deletion. In granule cells of wild-type animals, a wide range of single-channel conductances were observed (19-60 pS). The disruption of the NR2C gene results in the disappearance of low-conductance NMDA receptor channels ( < 37 pS) normally expressed in granule cells during developmental maturation. The NMDA receptor-mediated synaptic current is markedly potentiated in amplitude, but abbreviated in duration (with no net difference in total charge), and the non-NMDA component of the synaptic current was reduced. We conclude that the NR2C subunit contributes to functional heteromeric NMDA receptor-subunit assemblies at the mossy fiber synapse and extrasynaptic sites during maturation, and the conductance level exhibited by a given receptor macromolecule may reflect the stochiometry of subunit composition.

Fig. 2.

Whole-cell recording of spontaneous NMDA receptor channels in mature wild-type granule cells. A, Representative records of spontaneous NMDA receptor-mediated currents in a mature wild-type mouse granule cell. Two channel opening events with distinct unitary conductance and gating kinetics are clearly resolvable. B, Dwell-time distribution of open times with the fit of a double exponential (solid line) superimposed.C, Amplitude–frequency histogram with the fit of a double Gaussian distribution superimposed. Both conductances have extrapolated reversal potentials near 0 mV (D) and have corresponding conductances of 42 and 31 pS. All data were recorded in the same granule cell voltage-clamped at −60 mV in the absence of extracellular Mg²⁺.

Fig. 5.

Whole-cell recordings of spontaneous NMDA receptor channel activity in mature NR2C −/− granule cells are made up exclusively of large conductance events. A, A representative recording from a P16 −/− mouse granule cell at −60 mV. The open time (B, dwell time) and amplitude–frequency (C) distributions were fit by single-component functions (m = 43 pS; τ = 2.9 msec). D, The single-channel currents displayed linear I–Vrelations and reversed near 0 mV.

Fig. 7.

Synaptically activated single NMDA receptor channels of NR2C −/− granule cells are made up exclusively of high-conductance events. A, In the presence of nonsaturating concentrations of the competitive NMDA receptor antagonistd-AP5, the pharmacologically isolated NMDA receptor-mediated mossy fiber-evoked EPSC (i) (macroscopic EPSC recorded before the application of d-AP5) is made up of a flurry of directly resolvable single-channel openings (ii–iv). These channels closely resemble the spontaneously active NMDA receptor channels (B,C, open circles), as well as the channels activated by NMDA in an excised patch from the same cell (B,C, filled circles) and do not appear to express any small-conductance openings. The complete lack of small-conductance openings at any cellular locus argues against a nonspecific effect of localization. The similarity of synaptic and somatic channels lends credence to inferences about synaptic currents based on somatic receptors in preparations in which synaptic receptors are not as accessible.

Fig. 1.

Targeted disruption of the NR2C gene.A, Schematic representations of the targeting vector, wild-type allele, mutant allele, and the strategy for NR2C knockout. Four putative segments (M1–M4) are shown as open boxes. The location of the 5′- and 3′-external probes is shown. Both probes hybridize to a 15 kb EcoRI fragment from the wild-type allele and to 4.5 and 10.5 kb EcoRI fragments from the mutated allele, respectively. B, Southern blot analysis of progeny from intercross of heterozygotes (NMDA 2C +/−). 5′-External probe was used in this experiment. A similar result was obtained using the 3′-external probe (data not shown). C, Northern blot analysis of wild-type and mutated mice. Poly(A⁺) RNA was isolated from total brain tissue from 4-d-old animals. RNA was separated in a formaldehyde gel and transferred to a nylon membrane (Hybond-N+, Amersham). Membrane was hybridized with a fragment of NR2C cDNA containing all four transmembrane segments. The same filter was rehybridized with a β-actin probe.

Fig. 3.

NMDA receptor channels in excised outside-out patches from mature wild-type granule cells. Lefttraces illustrate representative examples of the conductance levels displayed by NMDA-activated channels in excised patches from four wild-type granule cells. Right graphs illustrate the amplitude–frequency histogram of a larger data set obtained from the patch shown to the left. Conductance levels in picosiemens for each patch reflect the estimated conductance after subtraction of junction potentials and estimation of the reversal potential (as in Fig. 2D).

Fig. 4.

The distribution of conductance states expressed by −/− mice is very similar to that observed in young rats.A, The proportional distribution of measured conductance levels in mature (>P14) wild-type mouse granule cells. B, The proportional distribution of measured conductance levels in mature (>P14) homozygous mutant (−/−) mouse granule cells (filled columns) and those measured in young (<P12) rat granule cells. All three groups show similar distributions of conductances of >39 pS. There is a marked difference in the distribution of conductances of <39 pS. The −/− mice appear to more closely resemble the young wild types and completely lack the low-conductance NMDA receptor channel.

Fig. 6.

NMDA receptor channels in excised outside-out patches from mature NR2C −/− granule cells lack low-conductance events. A, Single-channel currents activated by NMDA (20 μm) applied to a patch excised from a P15 NR2C −/− granule cell. Like the spontaneous channel openings observed in whole-cell recordings (Fig. 5), the NMDA-activated single-channel currents are made up exclusively of channels of large conductance (B) and long mean open time (C).

Fig. 8.

Mossy fiber-evoked macroscopic EPSCs in wild-type (+/+) and NR2C −/− granule cells. A, B, The composite mossy fiber-evoked EPSCs of granule cells in wild-type (+/+) and NR2C-deficient (−/−) mice recorded under control conditions (Control) and in the presence of CNQX (10 μm) and glycine (10 μm) to pharmacologically isolate the NMDA receptor-mediated component.C, The NMDA receptor-mediated components from the cells illustrated in A and B are superimposed, with the relative amplitudes vertically scaled to the mean observed in all cells (Table 1A) to illustrate the similarity of the net charge in the two cell types. D, The NMDA receptor-mediated currents from the cells illustrated in A and B are superimposed with their amplitudes scaled to the same peak, to illustrate the faster rate of decay of the synaptic current in granule cells from NR2C −/− mice.

Fig. 9.

Properties of the mossy fiber-evoked EPSC in homozygous mutant mouse granule cells. A, The mossy fiber-evoked composite EPSC under control conditions [−60 mV, 0 Mg²⁺, bicuculline (10 mm)]. The AMPA receptor- and NMDA receptor-mediated components of the composite EPSC can be resolved independently by the application of d-AP5 (50 μm) to illustrate the fast, AMPA receptor-mediated component (B), or the application of CNQX (10 μm) and glycine (10 μm) after recovery fromd-AP5 to illustrate the NMDA receptor-mediated component (C). Insets in B andC are faster time-base records with the control trace overlaid. All traces are from the same NR2C −/− mouse granule cell (P17). In D, the pharmacologically isolated NMDA receptor-mediated EPSC is displayed at a slightly higher gain and slower time base to display the biexponential decay of the EPSC; fit is overlaid as a solid line.