NMDA receptor antagonists reveal age-dependent differences in the properties of visual cortical plasticity

Abstract

The suggestion that NMDA receptor (NMDAR)-dependent plasticity is subunit specific, with NR2B-types required for long-term depression (LTD) and NR2A-types critical for the induction of long-term potentiation (LTP), has generated much attention and considerable debate. By investigating the suggested subunit-specific roles of NMDARs in the mouse primary visual cortex over development, we report several important findings that clarify the roles of NMDAR subtypes in synaptic plasticity. We observed that LTD was not attenuated by application of ifenprodil, an NR2B-type antagonist, or NVP-AAM007, a less selective NR2A-type antagonist. However, we were surprised that NVP-AAM007 completely blocked adult LTP (postnatal day (P) 45-90), while only modestly affecting juvenile LTP (P21-28). To assess whether this developmental transition reflected an increasing role for NR2A-type receptors with maturity, we characterized the specificity of NVP-AAM007. We found not only that NVP-AAM007 lacks discernable subunit specificity but also that the effects of NVP-AAM077 on LTP could be mimicked using subsaturating concentrations of APV, a global NMDAR antagonist. These results indicate that the effects of NVP-AAM077 on synaptic plasticity are largely explained by nonspecific blockade of NMDARs. Moreover our findings are the first to reveal a developmental increase in the sensitivity of LTP to NMDAR antagonism. We suggest that discrepant reports describing the effect of NVP-AAM077 on LTP may be partially explained by this developmental shift in the properties of LTP. These results indicate that the degree of NMDAR activation required for LTP increases with development, providing insight into a novel underlying mechanism governing the properties of synaptic plasticity.

FIG. 1.

The functional expression of synaptic NR2A/NR2B increases over development in the mouse visual cortex. A: postsynaptic density fractions, enriched for synaptic proteins, show the relative expression of NR2A and NR2B at 4 developmental time points. Samples were obtained by pooling together visual cortices from 3 to 10 brains (see methods). B: N-methyl-d-aspartate receptor (NMDAR) subunit expression normalized to P8 values. NR2A expression increases dramatically over development, whereas there is only a modest developmental increase in NR2B expression. Note that some error bars are obscured by the overlying symbol. C: shortening of NMDAR current durations over development reflects an increase in the relative level of NR2A expression. Using whole cell voltage-clamp recordings, NMDAR currents were isolated at 3 age groups from L2/3 pyramidal cells following extracellular L4 stimulation. Cells were held at +40 mV in the presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX; see methods). ○, averages of individual cells; •, the age group's average ± SE. A double-exponential fit of the deactivation current revealed a decrease in τ_w, corresponding to faster current kinetics over development. Stimulation artifacts were blanked in this and all subsequent figures for clarity.

FIG. 2.

NVP-AAM077 and ifenprodil have developmentally specific effects on the expression of low-frequency stimulation long-term depression (LFS-LTD) in the visual cortex. LTD was induced with 1-Hz stimulation (15 min) of L4 (↓). Data represent averages ± SE. Representative waveforms show field potentials before and after 1-Hz stimulation under control conditions. A: in young animals (P12-18), LTD is insensitive to either 3 μM ifenprodil or 50 nM NVP-AAM077. B: in juvenile animals (P21-28), LTD is modestly attenuated by either 3 μM ifenprodil or 50 nM NVP-AAM077. C: in adult animals (P45-90), only a small level of LTD could be induced by 1-Hz stimulation under control conditions.

FIG. 3.

Chemically induced LTD (chem-LTD) is insensitive to ifenprodil application. A: the magnitude of chem-LTD induced by brief application of NMDA (20 μM) is greater in the visual cortex of young (P12-18) animals than adult (P45-90) animals. Representative traces are shown from a P26 recording. B: ifenprodil (3 μM) fails to reduce the magnitude of chem-LTD, whereas 2-amino-5-phosphonovaleric acid (APV, 100 μM) prevents its induction.

FIG. 4.

The ability of ifenprodil or NVP-AAM077 to block LTP in the visual cortex is age-dependent. A: in juvenile animals (P21-28), LTP is significantly attenuated by either ifenprodil or NVP-AAM077. B: in adult animals (P45-90), 50 nM NVP-AAM077 completely blocks LTP, whereas 3 μM ifenprodil has no effect on its magnitude. In both panels, the traces shown above each plot are averages of the 15 min baseline and the last 15 min poststimulation of a representative recording under control conditions.

FIG. 5.

Unlike ifenprodil, NVP-AAM077 fails to alter NMDAR decay kinetics in a manner consistent with subtype specificity. A: perfusion of ifenprodil results in faster NMDAR current kinetics early in development (P21–P28), reflected as a decrease in τ_w, suggesting ifenprodil selectively blocks long-duration currents from NR2B-containing NMDARs. ○, raw data points; •, means ± SE before and after drug application. Right: traces are representative of NMDAR currents before and after ifenprodil application. *P < 0.05 from baseline. B: ifenprodil does not alter NMDAR decay kinetics in pyramidal cells from older mice (P45–P90), likely reflecting the small proportion of NR2B-containing NMDARs at this age. C: NVP-AAM077 fails to alter NMDAR current decay kinetics in cells from young mice. D: NVP-AAM077 also fails to alter NMDAR decay kinetics in older mice (P45–P90), when NR2A-containing NMDARs are expected to predominate at the synapse, suggesting that the drug lacks specificity for NR2A-containing NMDARs. E: data are normalized and summarized.

FIG. 6.

Block of NMDAR currents by ifenprodil, but not NVP-AAM077, correlates to developmental changes in NR2A/NR2B expression. A: antagonism of NMDAR currents by ifenprodil is reduced over development, reflecting the developmental decrease in relative NR2B expression. B: NVP-AAM077 similarly antagonized NMDAR currents over the same developmental time frame as the expression of NR2A increased. NVP-AAM077 also blocked a significant proportion of the NMDAR current in mice lacking NR2A. Taken together these data indicate that NVP-AAM077 lacks the ability to discriminate between NR2A and NR2B types in the mouse visual cortex.

FIG. 7.

There is a developmental increase in the ability to disrupt LTP with a nonsubunit-selective NMDAR antagonist. A: in juvenile (P21-28) and adult (P45-P90) mice, subsaturating concentrations of APV (1 μM) and NVP-AAM077 (50 nM) block a similar degree of NMDAR current amplitude. NVP-AAM077 data are replotted from Fig. 6B for the purposes of comparison. B: subsaturating APV fails to attenuate the induction of LTP in the visual cortex of young mice (P21-28). C: subsaturating APV dramatically reduces the magnitude of LTP in adult mice (P45-90).

FIG. 8.

There are no apparent differences between juveniles (P21-28) and adults (P45-90) in the L4-L2/3 field potential responses generated by 100-Hz stimulation. Example field potential traces from juvenile (A) and adult (B) stages of development. The first 28 pulses of a 100-Hz stimulus are depicted, and stimulus artifacts are clipped for clarity. C: the normalized field potential amplitude of the first 28 pulses of a 100-Hz stimulus does not differ between juvenile and adult animals.