Enhanced NR2A Subunit Expression and Decreased NMDA Receptor Decay Time at the Onset of Ocular Dominance Plasticity in the Ferret

Elizabeth B. Roberts and Ary S. Ramoa

Department of Anatomy, Visual/Motor Neuroscience Division, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0709


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Roberts, Elizabeth B. and Ary S. Ramoa. Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret. The NMDA subtype of glutamate receptor is known to exhibit marked changes in subunit composition and functional properties during neural development. The prevailing idea is that NMDA receptor-mediated synaptic responses decrease in duration after the peak of cortical plasticity in rodents. Accordingly, it is believed that shortening of the NMDA receptor-mediated current underlies the developmental reduction of ocular dominance plasticity. However, some previous evidence actually suggests that the duration of NMDA receptor currents decreases before the peak of plasticity. In the present study, we have examined the time course of NMDA receptor changes and how they correlate with the critical period of ocular dominance plasticity in the visual cortex of a highly binocular animal, the ferret. The expression of NMDA receptor subunits NR1, NR2A, and NR2B was examined in animals ranging in age from postnatal day 16 to adult using Western blotting. Functional properties of NMDA receptors in layer IV cortical neurons were studied using whole cell patch-clamp techniques in an in vitro slice preparation of ferret primary visual cortex. We observed a remarkable increase in NR1 and NR2A, but not NR2B, expression after eye opening. The NMDA receptor-mediated synaptic currents showed an abrupt decrease in decay time concurrent with the increase in NR2A subunit expression. Importantly, these changes occurred in parallel with increased ocular dominance plasticity reported in the ferret. In conclusion, molecular changes leading to decreased duration of the NMDA receptor excitatory postsynaptic current may be a requirement for the onset, rather than the end, of the critical period of ocular dominance plasticity.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Interruption of the visual input to one eye during a critical period of development leads to the loss of connections relaying information from the deprived eye to the visual cortex (Wiesel and Hubel 1965). In recent years, it has been proposed that the N-methyl-D-aspartate (NMDA) type of glutamate receptor plays a major role in this type of neuronal plasticity. Consistent with this hypothesis, blocking NMDA receptors with pharmacological agents (Bear et al. 1990; Rauschecker et al. 1990) or reducing NMDA receptor function using antisense DNA (Roberts et al. 1998) prevents the loss of responses to the deprived eye following monocular deprivation. However, the specific contribution of the NMDA receptor to ocular dominance plasticity has not been characterized. One approach to defining this role is to relate the time course of ocular dominance plasticity to developmental changes in the NMDA receptor. If the NMDA receptor plays an instructive role in ocular dominance plasticity, one expects some change in NMDA receptor properties coincident with the beginning and/or the end of the critical period.

In the neocortex, the NMDA receptor is composed of the NR1 subunit, which is required for receptor function, and the NR2A and NR2B subunits, which modulate NMDA receptor function (Laurie et al. 1997; Monyer et al. 1994; Watanabe et al. 1993; Wenzel et al. 1995; Zhong et al. 1995). All of these subunits have been shown to change expression patterns during early development in the rat (Monyer et al. 1994; Portera-Cailliau et al. 1996; Sheng et al. 1994; Zhong et al. 1995). Age-related changes in the expression pattern of NMDA receptor subunits may in turn underlie developmental changes in the functional properties, and especially kinetic properties, of cortical NMDA receptors (Flint et al. 1997; Monyer et al. 1992; Sheng et al. 1994; Takahashi et al. 1996; Vicini et al. 1998). The prevailing idea is that NMDA receptor-mediated synaptic responses decrease in duration after the peak of ocular dominance plasticity in rodents (Carmignoto and Vicini 1992; Flint et al. 1997). Accordingly, it is believed that shortening of the NMDA-receptor-mediated current underlies the developmental reduction of ocular dominance plasticity.

To understand the role of NMDA receptors in ocular dominance plasticity, it is now essential to examine how ontogenetic changes in the receptor correlate with the critical period of plasticity in the visual cortex of a highly binocular animal. Previous studies were based on experiments conducted in rodents, which are not highly binocular animals. In contrast, ferrets are known to have a considerable degree of binocular vision (Law et al. 1988). They provide, therefore, an ideal model to study the effects of monocular deprivation on cortical ocular dominance (Chapman et al. 1996) For this reason, we have examined how NMDA receptor kinetics and expression of NMDA receptor subunits correlate with visual development in the ferret. Our results indicate that major changes in NMDA receptor-mediated currents and molecular composition of the NMDA receptor occur at the onset, rather than the end, of the critical period of ocular dominance plasticity in the ferret.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Western blotting

Ferrets were deeply anesthetized with intraperitoneal pentobarbital (100 mg/kg) and killed by decapitation according to procedures approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University. The primary visual cortex was dissected out and protein samples were prepared as described previously (Roberts et al. 1998). Cortical protein samples (5 µg per lane) were separated by size using gel electrophoresis with 10% polyacrylamide minigels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose. Detection of specific proteins on the nitrocellulose was performed as described in Roberts et al. (1998). Three primary antibodies were used: rabbit anti-NMDAR1, rabbit anti-NMDAR2A, and rabbit anti-NMDAR2B. All antibodies were affinity purified polyclonal antibodies (Chemicon, Temecula, CA); each was used at a dilution of 1:1,000. The secondary antibody was a goat anti-rabbit IgG (whole molecule) peroxidase-conjugated antibody (Sigma, St. Louis, MO) diluted 1:1,000. Band densities were measured by densitometry with an image analysis unit (Imaging Research, St. Catherine's, Ontario) using the MCID/M4 image analysis software. Pixel intensities were converted to optical densities by using a density wedge. Background optical density was subtracted from all band density measurements. All optical densities were normalized to the optical density of the P35 sample; a sample of this age was present in all immunoblots. This normalization allowed band densities from different immunoblots probed with the same antibody to be compared.

Electrophysiological recordings

Animals were killed with an overdose of intraperitoneal pentobarbital (100 mg/kg). Slices of visual cortices were prepared as described previously (Blanton et al. 1989; Ramoa and McCormick 1994a). Slices were maintained at room temperature and superfused with buffered and oxygenated solution containing (in mM) 126 NaCl, 2.5 KCl, 2 MgSO4, 26 NaHCO3, 1.25 NaHPO4, 2 CaCl2, and 10 dextrose, saturated with 95% O2-5% CO2 to a final pH of 7.4. To activate synaptic input to cortical cells, constant current stimuli (100-µs, 30- to 300-µA intensity) were delivered through a bipolar stimulating electrode positioned in the cortical white matter. Whole cell recordings were obtained using the patch-clamp technique as described previously (Blanton et al. 1989; Ramoa and McCormick 1994a). Recordings of NMDA receptor-mediated currents were conducted at +40 mV in the presence of bicuculline methiodide (30 µM) and 6-nitro-7sulfamoylbenzo[f]-quinoxaline-2,3-dione (NBQX; 10 µM) to block GABAA and alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, respectively. To study the AMPA receptor-mediated currents, NBQX was omitted from the perfusate and recordings were conducted at -70 mV. Patch electrodes (4-8 MOmega ) were filled with the following solution (in mM): 120 cesium gluconate, 10 NaCl, 10 HEPES buffer, 1 sodium EGTA, 2 MgCl2, 0.1 CaCl2, 2 Na-ATP, and 0.1 Na-GTP, maintained at pH 7.25. Recordings were obtained using an Axopatch-1D amplifier, and the pClamp data acquisition program (Axon Instruments, Foster City, CA) was used to acquire and analyze data.


    RESULTS
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INTRODUCTION
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DISCUSSION
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The expression of NMDA receptor subunits NR1, NR2A, and NR2B was examined in the primary visual cortices of 19 ferrets ranging in age from P16 to adult (n = 2-4 animals and 4-8 hemispheres for each age) using Western blotting. Functional properties of NMDA and AMPA receptors were studied in 51 layer IV neurons using whole cell patch-clamp techniques. The ages of animals used for electrophysiological recordings ranged from P21 to P44.

Increased NR2A subunit expression occurred during the increase in visual plasticity

Developmental changes in NR1, NR2A, and NR2B subunit expression were examined using specific antibodies in Western blotting. The results in Fig. 1 illustrate the finding that both NR1 and NR2A subunit expression increased substantially around the time of eye opening (approximately P32) and reached a peak around P45. These ages correspond to the onset and peak, respectively, of ocular dominance plasticity in the ferret (N. P. Issa, J. T. Trachtenberg, B. Chapman, and M. P. Stryker, personal communication). NR2B expression, in contrast, remained elevated through early development and into the critical period. All subunits exhibited a marked decrease in expression from the critical period to adulthood.



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Fig. 1. NMDA receptor subunit expression in ferret visual cortex during development. Western blots containing total protein samples from the visual cortices of ferrets ranging in age from P16 to adult were probed with polyclonal antibodies against NR1, NR2A, and NR2B subunits. Age of the animal from which each sample was derived is indicated above the blots; the numbers indicate postnatal age in days.

Quantification of the Western blotting results was obtained by determining relative optical densities (ratios of band optical densities at each age divided by the density observed in the same immunoblot at P35) for four to eight visual cortical samples at each age. Means and standard errors of the relative optical densities at each age are shown in the bar graphs of Fig. 2. The graphs indicate that NR1 and NR2A expression increased significantly (P <=  0.05, 1-way ANOVA followed by Tukey's test) from P23 to P35. Expression of both subunits remained high through P45 then declined into adulthood. The decline in NR1, but not NR2A, expression is significant (P < 0.05) from the critical period to adulthood. There is also a significant (P < 0.05) decline in NR2B expression after the end of the critical period. These changes in subunit expression most likely reflect alterations in the ratio of NR2A and NR2B subunits within individual receptors (Luo et al. 1997). In conclusion, our results indicate that early in postnatal development, the ratio of NR2B/NR2A is high in ferret visual cortex. After eye opening, there is a decrease in the NR2B/NR2A ratio as the expression of the NR2A subunit increases, whereas NR2B expression remains relatively constant.



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Fig. 2. Quantification of NR2A, NR2B, and NR1 protein levels in ferret visual cortex. Relative optical densities are the ratios of band densities at each age divided by the band density of the P35 sample from the same immunoblot. Means ± SE of the ratios are graphed vs. postnatal age (n = 4-8 hemispheres for each age).

Decay time of NMDA-EPSCs decreased markedly at the onset of the critical period

We have examined whether the changes in subunit composition described earlier are temporally related to changes in NMDA receptor function. Whole cell patch-clamp recordings from visual cortical neurons revealed a significant (P < 0.05, independent t-test, n = 23 cells before P32 and n = 11 after P32) age-related reduction in NMDA-excitatory postsynaptic current (EPSC) decay time after eye opening, as shown in the examples of Fig. 3A. The scatter plot shows that abrupt changes in kinetic properties occurred after eye opening, a time when NR1 and NR2A subunit expression increased. Kinetic properties of AMPA-EPSCs did not change significantly during this period (P > 0.05, independent t-test, n = 9 cells before P32 and n = 9 cells after P32) as illustrated in Fig. 3B. These results indicate that developmental changes in diffusion or uptake of glutamate that would have affected NMDA and AMPA receptors similarly did not determine the changes in NMDA-EPSCs. Rather, changes in subunit composition may underlie the modifications in the NMDA-EPSC of visual cortical neurons.



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Fig. 3. Changes in NMDA receptor-mediated current decay times in layer IV visual cortical cells. Examples of an NMDA-excitatory postsynaptic current (EPSC; A) and an alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-EPSC (B; averaged responses from 10 trials) stimulated through cortical afferents. Cells studied are from ferrets before eye opening (P26-P29) and after eye opening (P37-P38). Decay constants (tau ) shown are means ± SE. C: scatter plot of decay times vs. postnatal age for 33 layer IV visual cortical cells. Bar shows the relationship of the critical period of visual plasticity to changes in NMDA-EPSC decay time.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this report we have examined developmental changes in subunit composition and functional properties of the NMDA receptor in the ferret primary visual cortex. We found an increase in the expression of NR2A and NR1 subunits that was temporally correlated with a decrease in NMDA-EPSC decay time. Both changes occurred after eye opening around P32. To understand the role of NMDA receptors in plasticity, it is important to examine how these changes correlate with the critical period of ocular dominance plasticity, which has been well characterized in the ferret. Sensitivity to the effects of monocular deprivation starts after P30, is greatest around P45, and ends around P70 in ferrets (N. P. Issa, J. T. Trachtenberg, B. Chapman, and M. P. Stryker, personal communication). Therefore our results indicate a remarkable correlation between shortening of the decay time of the cortical NMDA-EPSC, increased expression of NR2A and NR1 subunits, and increased ocular dominance plasticity in ferret visual cortex.

Previous studies have shown that NMDA receptor-mediated synaptic transmission is enhanced markedly in several neural structures during early development (Fox et al. 1989; Hestrin 1992; Ramoa and McCormick 1994b; Tsumoto et al. 1987). This enhancement has been proposed to result from an increase in the number of NMDA receptors (Bode-Greuel and Singer 1989) and from a prolonged duration of NMDA-EPSCs in young animals relative to adults (Carmignoto and Vicini 1992; Hestrin 1992; Ramoa and McCormick 1994b; Ramoa and Prusky 1997). The increased number of receptors is reflected in an increase in the expression of NR1 subunits (Catalano et al. 1997) and, as shown here, NR2A subunits.

Carmignoto and Vicini (1992) reported that the duration of NMDA-EPSCs in rat visual cortex was quite long in early development and became progressively shorter with age. Their conclusion was that a prolonged duration of NMDA-EPSCs may be required for visual cortical plasticity, whereas later shortening of the open time leads to a decline in plasticity. However, this conclusion is not consistent with our finding that NR2A subunits are expressed at the beginning of the critical period of ocular dominance plasticity. Enhanced expression of the NR2A subunits has been shown to result in NMDA receptors with relatively shorter decay kinetics (Flint et al. 1997; Monyer et al. 1992; Vicini et al. 1998). Therefore the developmental profile of NR2A subunit expression in the ferret provides support for our proposal that fast, rather than slow, kinetics are present at the onset of ocular dominance plasticity. Interestingly, some of Carmignoto and Vicini's (1992) experimental results are in agreement with our proposal. In their study, the contribution of the slow component to NMDA-EPSCs in rat visual cortex decreased throughout the first postnatal month, reaching adult levels as early as P30 (Carmignoto and Vicini 1992, Fig. 1C), a time when ocular dominance plasticity is at a peak in rats (Fagiolini et al. 1994). Moreover, Flint et al. (1997) have shown increased NR2A subunit expression and changes in NMDA-EPSC kinetics at relatively early stages of development. In their defense, Carmignoto and Vicini (1992) published their results before the critical period of visual plasticity in rats was clearly defined (Fagiolini et al. 1994).

The idea that slow NMDA receptor kinetics are necessary for visual plasticity poses an important conceptual problem when considering the role of NMDA receptors in Hebbian mechanisms of neuronal plasticity (Rauschecker 1991). Long-duration NMDA-EPSCs would be disruptive to ocular dominance plasticity because during monocular deprivation, strong signals from the normal eye as well as weak inputs from the deprived eye would be detected as coincident even when they are separated by several hundred milliseconds. According to Hebbian mechanisms (Hebb 1949), this would lead to the stabilization of inputs from the deprived eye. This is contrary to the experimental result in which inputs from the deprived eye are lost (Wiesel and Hubel 1965).

Our interpretation that major changes in NMDA receptors take place at the beginning of the critical period of ocular dominance plasticity is contrary to much of the literature. However, several of our experimental results agree with previous studies. How can this discrepancy be explained? One reason is that there are few species in which the time course of the critical period has been defined clearly. Age-related NMDA receptor changes in these species can, therefore, be difficult to relate to changes in ocular dominance plasticity. Another reason is that the present study gives a comprehensive measurement of the expression of relevant NMDA receptor subunits over the relevant time period in development while providing a careful measurement of the time course of changes in pharmacologically isolated postsynaptic events. In contrast, many earlier studies in the literature measured only one of these characteristics. The results of these reports are difficult to relate to one another.

It is important to point out that we have studied only one of several types of visual cortical plasticity. It has been shown that the development of orientation selectivity occurs most rapidly during the 2 weeks after eye opening in the ferret visual cortex (Chapman and Stryker 1993). Therefore one would expect there to be a high degree of plasticity of orientation selectivity during this same period. This is the period during which we have observed major changes in the NMDA receptor. Nonetheless until the critical periods for orientation and direction selectivity are determined in the ferret, we cannot assume that the same correlations with NMDA receptor changes apply to these types of plasticity.

In summary, kinetic properties of the NMDA receptor are determined mainly by the relative amounts of NR2A and NR2B subunits in the heteromeric NMDA receptor assembly (Monyer et al. 1992; Vicini et al. 1998). Our results indicate that before the critical period of ocular dominance plasticity when the ratio of NR2B/NR2A subunits is high, slow channel kinetics predominate. This results in NMDA-EPSCs that are long-lasting; this may be important in enhancing excitatory responses at a time when synapses are immature (Campbell and Shatz 1992), visual stimulation is not yet possible and low frequency waves of retinal activity predominate in visual pathways (Meister et al. 1991). However, after eye opening, the number of NMDA receptors increases markedly, coinciding with the onset of sensory stimulation. Long-lasting NMDA-EPSCs are, therefore, no longer required to enhance excitatory responses and actually may be detrimental to visual processing. We propose that for the critical period of ocular dominance plasticity to occur, subunit combinations with slow channel kinetics must be replaced by those with fast kinetics that can support the role of the NMDA receptor as a correlation detector in vision.


    ACKNOWLEDGMENTS

We thank N. Patel for contributing to the Western blotting experiments.

This work was supported by National Eye Institute Grant EY-11508, the National Science Foundation (IBN-9308576), and the Whitehall Foundation.


    FOOTNOTES

Address for reprint requests: A. S. Ramoa, Dept. of Anatomy, Virginia Commonwealth University, 1101 E. Marshall St., Box 0709, Richmond, VA 23298-0709.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 September 1998; accepted in final form 21 January 1999.


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