Laminar distribution of NMDA receptor subunit (NR1, NR2A, NR2B) expression during the critical period in cat visual cortex

Molecular Brain Research 119 (2003) 19 – 27 www.elsevier.com/locate/molbrainres

Research report

Laminar distribution of NMDA receptor subunit (NR1, NR2A, NR2B) expression during the critical period in cat visual cortex George D. Mower a,*, Lu Chen b a

Department of Anatomical Sciences and Neurobiology, Health Sciences Center, University of Louisville School of Medicine, 500 South Preston St., A Bldg., Rm. 902, Louisville, KY 40202, USA b Department of Ophthalmology and Visual Sciences, University of Louisville, School of Medicine, Louisville, KY 40202, USA Accepted 13 August 2003

Abstract Changes in NMDA subunit composition may be part of the molecular basis for critical period plasticity. The present study used immunohistochemistry to determine developmental changes in the laminar distribution of the three major cortical NMDA subunits (NR2A, NR2B, NR1) during the critical period in cat visual cortex. For all three subunits, at 1 week staining was concentrated in two bands: an upper band consisting of layer I, the compact zone and the upper half of the cortical plate; a lower band consisting of layers V and VI. In the lower part of the cortical plate (immature layer IV) staining was very low. For NR2A and NR2B, immunoreactivity in layer IV remained low until 10 weeks of age. At 20 weeks and adult, layer IV filled in and NR2A and NR2B label was rather uniform across all layers. NR1 showed a developmental pattern of expression different from NR2A and NR2B after 1 week. At 5 and 10 weeks, label was prominent in layer IV and superficial layers, but low in layers V and VI. The main change after 10 weeks was a progressive decrease in staining, such that in older animals label was markedly densest in superficial layers. Thus, during the rise of the critical period, NR1 is the dominant subtype in layer IV and could play a role in anatomical ocular dominance column formation and plasticity. At the same time, NR2A and NR2B subunits are concentrated outside layer IV, and could be related to physiological plasticity in extragranular layers, which precedes and outlasts plasticity in layer IV. For all three NMDA receptor subunits, the laminar distribution was similar in normal and dark reared visual cortex at 20 weeks, indicating that the developmental changes in laminar pattern of expression are independent of visual input. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Visual system Keywords: Neuronal plasticity; Visual development; Neurotransmitter receptor

1. Introduction NMDA receptors, which mediate excitatory neurotransmission in central synapses, are multimeric proteins composed of NR1 and NR2 subunits. At least five subunits, NR1, NR2A, NR2B, NR2C and NR2D, have been cloned [33 – 35,45,46,48]. NR1 appears to be a mandatory subunit that is necessary for the major characteristic properties, including Ca2 + permeability, Zn2 + inhibition, Mg2 + blockade, agonist and antagonist selectivity and glycine modulation [46,52]. NR2A-2D confer functional variability to the

* Corresponding author. Tel.: +1-502-852-5177; fax: +1-502-8526228. E-mail address: [email protected] (G.D. Mower). 0169-328X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.08.007

receptor [46,52]. In the cerebral cortex, NR1, NR2A and NR2B are the dominant subunits [47,61]. In visual cortex, NMDA receptor activation has been implicated as critical for physiological plasticity during early postnatal life and blockade of these receptors reduces the shift in ocular dominance due to monocular deprivation during the critical period [3,17,59]. The visual cortex is an ideal model to study development and plasticity of NMDA receptor subunits because of its well-documented postnatal critical period for anatomical and physiological plasticity, as assessed by susceptibility to monocular deprivation. Plasticity in the visual cortex is absent until 3 weeks of age, peaks at about 5 weeks, gradually declines to low levels at 20 weeks and disappears at about 1 year of age [15]. Additionally, the time course of the critical period can be extended by rearing cats in total darkness [12,50]. Devel-

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opmentally, changes in the relative level of expression of NR1, NR2A and NR2B during the visual cortical critical period have been described in rodents, ferrets and cats [9,47,55,56,58,61]. In parallel, there are decreases in both the NMDA-mediated component of visually evoked responses [13,23] and in the duration of NMDA-mediated synaptic currents [6,29,58]. Changes in NMDA receptor subunit composition have been implicated as a mechanism for these changes in NMDA synaptic efficacy. For example, insertion of NR2A receptors has been shown to correspond to the shortened duration of NMDA currents [22,58,66] and thus the transfer of the receptor from an immature to a mature kinetic type. Originally, the changes in NMDA receptor currents and subunit composition were proposed to correlate with the elimination of visual cortical plasticity in rats [6]. When the time course of the rat visual cortical critical period was determined [21], however, it became clear that the change to adult duration NMDA currents occurs near the peak of the critical period, a finding that has been directly confirmed in ferrets [58]. Analysis of the expression of NMDA receptors in ferrets and cats indicated that levels of all three subunits are maximal at the peak of plasticity and decline thereafter [9,58]. In rats, the ratio NR2A/NR2B was found to rise and then decline in parallel with the critical period [55,56]. Thus, changes in NMDA receptor subunit composition and receptor currents appear more associated with the presence of critical period plasticity than with its termination [44]. The degree of plasticity across visual cortical laminae differs as a function of age. For example, in cat visual cortex, plasticity persists until later ages in extragranular layers than it does in layer IV [16,51]. Therefore, an important issue for understanding the role of NMDA receptor subunits in the visual cortical critical period is the laminar pattern of expression of the different subunits across development. The present study was undertaken to determine laminar changes in the expression of NMDA subunits NR1, NR2A and NR2B in cat visual cortex. Additionally, since the NR2A subunit was shown to be selectively affected by prolonged dark rearing [9], we compared the laminar distribution of NMDA subunits in normal and dark reared cats.

(0.9% NaCl in 0.1 phosphate buffer, pH = 7.4) followed by 4% paraformaldehyde in PBS. The brains were then immersed in 30% sucrose in PBS at 4 jC until they sank. Blocks of the brains were frozen on dry ice and stored at 80 jC until use. For Western blot studies, fresh brain regions (all of area 17 and possibly a small part of 18) were dissected from two of the adult cats, immediately frozen in liquid nitrogen, and stored at 80 jC until use. 2.2. Tissue preparation For Western blots, a subcellular fraction containing crude synaptic membranes was prepared for Western blotting with electrochemoluminescent detection as described previously [9,27]. For immunohistochemistry study, visual cortex was sectioned coronally at 30 Am on a cryostat (Cryocut 1800, Reichert-Jung, Austria). 2.3. Antibodies For immunohistochemistry studies, a monoclonal antibody for NR1 (1:1000 dilution) and a polyclonal antibody specific to NR2B (1:100) were from Chemicon International. A polyclonal antibody specific to NR2A (1:250) was from Upstate Biotechnology. The specificity of each antibody was determined by Western blotting analysis as shown in Fig. 1. Each antibody labeled a single band of appropriate molecular weight (NR2A: 180 kDa, NR2B: 180 kDa, NR1: 120 kDa). Rabbit IgG and Mouse IgG ABC Kits were from Vector Laboratories (Burlingame, CA). 2.4. Immunohistochemistry Free-floating sections were first immersed in blocking solution (10% serum, 0.2% Triton-X in phosphate-buffered saline [PBS]) for 1 h at room temperature (RT) followed by primary antibody incubation for 2.5 days at 4 jC. The sections were then thoroughly rinsed in PBS and incubated in biotinylated secondary antibody (1:200 in PBS) for 1 h at RT. After incubation with avidin– biotin

2. Materials and methods 2.1. Animals A total of 19 cats were used for this study. They were reared in a normal 12 light/dark cycle until 1, 5, 10, 20 weeks (n = 3 at each age) or adult (n = 5) or in complete darkness from birth to 20 weeks of age (n = 2). The animals were killed by an overdose of sodium pentobarbital (75 mg/kg i.p.). These procedures conform to the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee. For immunohistochemical studies, perfusion was performed intracardially with PBS

Fig. 1. Western blots showing the specificity of the antibodies to NMDA receptor subunits (NR2A, NR2B, NR1) in adult cat visual cortex. Each antibody labeled a single band of appropriate molecular weight (NR2A: 180 kDa, NR2B: 180 kDa, NR1: 120 kDa). Note that lines indicating 216, 132 and 78 kDa molecular size markers differ across the three separate blots.

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complex reagent (ABC kit, Vector Laboratories) for 1 h, labeling was visualized by treatment with diaminobenzidine (DAB, 0.7 mg/ml) and hydrogen peroxide (0.2 mg/ ml) in Tris buffer (0.06 M). Sections were then washed and mounted onto subbed slides and air dried overnight. Sequential dehydration (through 50%, 70%, 95% and 100% alcohol) and clearance (with xylene) were performed before the slides were cover-slipped with Permount (Fisher). Adjacent sections were counterstained with cresyl violet to identify cortex layers. Control sections were identically processed but with the primary antibody replaced by normal serum. No staining was present in these sections. Cats at each age and rearing condition were processed together in each immunohistological experiment. Immunohistochemistry was repeated on each animal to ensure reproducibility of the results. The slides were photographed using a digital system (SPOT software, v2.2, SPOT Diagnostic Instruments). Images were adjusted for consistent brightness and contrast across ages and subunits. The analysis focused on laminar differences in immunostaining and no analysis of comparative levels of expression is reported. Differences in the level of expression of NMDA subunits were determined previously by Western blots of crude synaptic fractions [9].

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intensity midway through the cortical mantle and a band of intermediate intensity at the bottom of the cortical mantle. Outside of area 17, the splenial sulcus ventrally and area 18 dorsally showed comparatively homogeneous label throughout all cortical layers for all three subunits. To analyze the immunoreactivity in neonatal (1 week) cat VC, it is necessary to account for the immature lamination pattern at this age. As shown in the leftmost panels of Figs. 4 –6, we followed the laminar designations of Luskin and Shatz [42,43]. In neonates (1 week), layers V and VI are identifiable and a significant number of cells in layer VI are remnants of the embryonic subplate. At this age, cells in the superficial cortical plate (CP) are destined for the layer III/ IV border in the adult and cells in the deep cortical plate are destined to become layer IV. The remaining cells that will form layers II and the rest of layer III are densely packed in the compact zone (CZ). Layer I, which corresponds to the embryonic marginal zone (MZ), is also identifiable. The bands of distinctly lower immunoreactivity for NMDA receptor subunits in Fig. 2, therefore, correspond to the developing layers IV and VI. Thus, in the neonatal visual cortex, NMDA receptor subunits are selectively reduced in expression in layers that are the principal recipients of geniculo-cortical afferents. 3.2. Intracellular distribution of NMDA subunit immunoreactivity

3. Results 3.1. Laminated expression of NMDA receptor subunits in neonatal area 17 Fig. 2 shows the laminar pattern of expression of NR1, NR2A and NR2B in consecutive sections from visual cortex of a 1-week-old cat. The low magnification photomicrographs of Fig. 2 show that for all three NMDA receptor subunits, area 17 was demarcated by reduced immunostaining in two parallel bands: a band with distinctly low

Each antibody labeled cell bodies, processes and neuropil, as shown in Fig. 3. The NR1 and the NR2B immunoreactivity in cell bodies was mainly in the cytoplasm and densest toward the cell membrane. The NR2A immunoreactivity in cell bodies was densest in the nucleus and lighter elsewhere. Each antibody also stained processes, as demonstrated by the apical dendrites of the layer V cells as well as the finer processes shown in Fig. 3. For each NMDA receptor subunit, the intracellular pattern of label was consistent at all ages.

Fig. 2. Low magnification photomicrographs of the laminar distribution of NMDA receptor subunit (NR2A, NR2B, NR1) immunoreactivity in consecutive sections from the visual cortex of 1-week-old cats. Arrowheads indicate boundaries of area 17. Two bands of low immunoreactivity are evident for each subunit. These bands correspond to the developing layers IV and VI (see text for details). Scale bar at bottom of rightmost panel indicates 500 Am.

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Fig. 3. High magnification photomicrographs showing the intracellular distribution in layer V pyramidal cells of immunoreactivity revealed by NMDA receptor subunit specific antibodies (NR2A, NR2B, NR1). Each antibody labeled cell bodies, dendrites and neuropil. For NR1 and NR2B, immunoreactivity in cell bodies was mainly in the cytoplasm and densest toward the cell membrane. For NR2A, there was dense staining of the nucleus. Scale bar at bottom of rightmost panel indicates 25 Am.

3.3. Developmental changes in NR2A and NR2B subunit expression Subunits NR2A and NR2B showed similar developmental changes in the laminar pattern of expression during and after the critical period, as shown in Figs. 4 and 5. At 5 weeks, the pattern seen at 1 week was maintained, with label being highest in layers I, II, III and V and much lower in layers IV and VI. Within layer IV, there was low cell and neuropil staining but the passing dendritic shafts of cells in other layers were clearly immunostained. At 10 weeks, somatic and neuropil staining became more evident in layers IV and VI for NR2A, but it was still much lower than that in the other layers. Immunostaining for NR2B continued to be low in cell bodies and neuropil in layers IV and VI at 10

weeks. At later ages, immunostaining for both NR2A and NR2B showed a relative increase in layers IV and VI such that by 20 weeks cellular staining was rather uniform across all cortical layers. There was a greater intensity of neuropil staining in superficial layers I– III for both subunits. This staining pattern for NR2A and NR2B across visual cortical layers persisted in adults. 3.4. Developmental changes in NR1 subunit expression Developmental changes in the laminar pattern of expression of NR1 differed markedly from those for NR2A and NR2B, as shown in Fig. 6. At 5 weeks, NR1 expression was highest in layers II, III and IV and low in layers I, V and VI. This laminar distribution continued

Fig. 4. Developmental changes in the laminar distribution of NR2A in cat visual cortex. Laminar boundaries (layers I – VI) were determined from adjacent cresyl violet stained sections. In 1-week-old animals, the laminar designations of Luskin and Shatz [42,43] were used (CZ: compact zone, CP: cortical plate, SP: subplate). In older cats, visual cortical layers I through VI are indicated. Scale bar at bottom of rightmost panel indicates 100 Am.

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Fig. 5. Developmental changes in the laminar distribution of NR2B in cat visual cortex. Laminar boundaries (layers I – VI) were determined from adjacent cresyl violet stained sections. In 1 week animals, the laminar designations of Luskin and Shatz [42,43] were used (CZ: compact zone, CP: cortical plate, SP: subplate). In older cats, visual cortical layers I through VI are indicated. Scale bar at bottom of rightmost panel indicates 100 Am.

at 10 weeks, with staining intensity in layer IV decreasing somewhat. At 20 weeks, there was a further reduction in NR1 staining intensity in layer IV and the highest levels of

expression were restricted to layers II and III. This staining pattern for NR1 across visual cortical layers persisted in adults.

Fig. 6. Developmental changes in the laminar distribution of NR1 in cat visual cortex. Laminar boundaries (layers I – VI) were determined from adjacent cresyl violet stained sections. In 1-week-old animals, the laminar designations of Luskin and Shatz [42,43] were used (CZ: compact zone, CP: cortical plate, SP: subplate). In older cats, visual cortical layers I through VI are indicated. Scale bar at bottom of rightmost panel indicates 100 Am.

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Fig. 7. Comparison of the laminar distribution of NR2A immunoreactivity in the visual cortex of 20-week-old normal and dark reared cats. Laminar boundaries (layers I – VI) were determined from adjacent cresyl violet stained sections are indicated. Scale bar at bottom of right panel indicates 100 Am.

3.5. Normal laminar pattern of NMDA receptor subunits in dark reared cat visual cortex Since each NMDA receptor subunit showed clear developmental changes in the laminar pattern of expression, an important question is whether these laminar changes depend on visual input. Of particular interest is the NR2A subunit because Western blot studies [9] showed that the level of this protein, but not that of NR1 or NR2B, is modified by dark rearing. As shown in Fig. 7, dark rearing for 20 weeks, which produces a maximal enhancement of physiological plasticity in the visual cortex of dark reared cats [49] and significantly elevates NR2A levels [9], did not prevent or alter developmental changes in the laminar pattern of NR2A expression. Similarly, no effect of prolonged dark rearing was found for NR1 or NR2B subunit expression across cortical layers (data not shown).

4. Discussion The present results provide the first comparative data on developmental changes in the laminar pattern of expression of the NR1, NR2A and NR2B subunits of the NMDA receptor in cat visual cortex. Prior to the onset of the critical period (1 week), all three subunits were preferentially expressed in layers II, III and V, whereas, the geniculocortical afferent recipient layers IV and VI showed low expression. During the critical period, the laminar changes

in the NR2A and NR2B expression were similar to each other, but both were nearly complementary to those of NR1. For NR2A and NR2B, low expression in layer IV and to a lesser extent in layer VI persisted past the peak of the critical period (5 weeks) until at least 10 weeks of age. For NR1, on the other hand, at 5 and 10 weeks expression was high in layer IV (in contrast to NR2A and NR2B) as well as the superficial layers, but low in layers V and VI. During the decline of the critical period (after 10 weeks), the main change for NR2A and NR2B was a filling in of laminar expression, and cell body staining became rather uniform across all cortical layers at 20 weeks and adult. Neuropil staining for NR2A and NR2B was highest in superficial layers at 20 weeks and adult. Conversely, for NR1 there was a progressive decrease in staining, such that in 20 week and adult animals expression was markedly highest in superficial layers II and III. These temporal differences in the laminar distribution of NMDA subunits are consistent with developmental changes in subunit protein levels [9] and suggest changing balances in the relative NR1:NR2 composition of the receptor. The NMDA receptor is believed to be a heterotetrameric structure with four subunits assembled around a central ionpermeable channel [38], but the possibilities that the receptor is pentameric (see Ref. [18], for review) and that there are functional NR1 homomers [25] have not been eliminated. While at least one NR1 subunit must be present to form a functional channel, the composition of the remaining subunits is variable. In the forebrain, there is evidence for the existence of both binary (NR1/NR2A and NR1/NR2B) and probably more abundant ternary (NR1/NR2A/NR2B) complexes [8,20,28,41,61]. NMDA receptor subunit proteins are expressed both at the post synaptic membrane of excitatory synapses and at extrasynaptic cell membrane and intracellular sites [20,53], suggesting differential targeting of distinct types of NMDA receptor assemblies between intracellular and post-synaptic sites based on subunit composition. Both lateral mobility of synaptic and extrasynaptic receptors within the membrane [63] as well as insertion into synapses of receptors from intracellular pools [26,36,37] have been demonstrated, and direct imaging in hippocampal slices has shown subunit specific trafficking of NR2A and NR2B [2]. Although the NMDA receptor is typically composed of NR1 plus NR2A and/or NR2B subunits, because of its biochemical complexity, the relative number of each subtype across individual receptors, and consequently the relative immunohistochemical staining of individual subunits, can differ widely. The differential laminar and intracellular distribution of NR1 and NR2 subunits as well as the developmental changes found here and in other studies [4,5] reflect the complex and dynamic structure of the NMDA receptor. The changes in the laminar pattern of expression of NMDA receptor subunits were independent of visual experience, since the laminar patterns were similar in normal and dark reared animals. Catalano et al. [7] also found no effect

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of dark rearing on the laminar pattern of NR1 expression in visual cortex. However, there are demonstrated effects of dark rearing on the relative subunit composition of the NR2A receptor in synaptosomal fractions [9,55,56], as well as physiological effects on NMDA receptor function [13,24,54]. Based on the available evidence, these effects do not appear to reflect an alteration of general developmental changes in subunit expression across cortical laminae, but finer histological and biochemical analyses at the subcellular level may reveal relevant effects of dark rearing. Changes in a particular subunit’s contribution to the NMDA receptor in dark reared animals could reflect either a generalized cellular change in level or a redistribution between synaptic and extrasynaptic sites.

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antibody as the present study, we reacted sections with a different NR2A antibody (Chemicom International). The same intracellular distribution resulted, indicating that the labeling pattern was not antibody specific. Additionally, Western blots with all of the subunit antibodies used in our immunohistochemistry [9] (present results) indicated that single bands of appropriate size were labeled by each. Perhaps the distinct intracellular labeling pattern of NR2A seen in paraformaldehyde-fixed tissue is related to the fact that NR2A, unlike NR1 and NR2B, is capable of being rapidly inserted into the membrane surface in response to visual input [55,56]. The epitopes identified by the antibodies to the three subunits could also be differentially accessible to binding in the various cellular compartments in fixed tissue.

4.1. Relation to previous work 4.2. Relation to critical period plasticity Developmental changes in the laminar pattern of expression of NR1 in cat visual cortex were described previously [7]. Although the time points sampled differ somewhat between that study and the present one, the patterns observed are quite similar. The present study found that NR1 expression in layers IV and VI is low prior to the critical period (1 week) and the data of Catalano et al. [7] indicate that this pattern persists at postnatal days 15 and 28. Both studies found high NR1 expression in layers II – IV with low levels in layers V and VI near the peak of the critical period (5 and 10 weeks in the present results, 40 and 53 postnatal days in Ref. [7]) and both studies found expression highest in superficial layers II and III in older cats. While there is good agreement between the present results and those of Catalano et al. [7], it is important to note that a study by Trepel et al. [65] found a patchy distribution of NR1 expression and markedly different laminar patterns during development of cat visual cortex. The antibodies used by Trepel et al. [65] and Catalano et al. [7] were the same and the reason for this discrepancy is unclear. Laminar patterns of expression of all three NMDA receptor subunits during postnatal development have been described in rats [4,5] and the results show a number of similarities with the present data in cats. In both species, developmental changes in NR2A and NR2B were similar to each other and these subunits showed different development regulation than NR1. Expression of all three subunits was highest superficial to layer IV in both species. In rats, there was not a clear distinction between layer IV and the other layers as seen in cats, a difference that may be related to the lack of ocular dominance columns in rodents [19]. The intracellular patterns of immunoreactivity for the subunits were also similar in the rat and cat, with NR2A showing dense nuclear label. As discussed above, both synaptic membrane and intracellular localization was expected for all subunits since all are expressed in widespread cellular compartments; nevertheless, the high intracellular concentration of NR2A was unexpected for a neurotransmitter receptor subunit. Since Cao et al. [4,5] used the same

The present results relate most directly to the two major laminar specific events that occur during the critical period: (1) the formation of anatomical ocular dominance columns within layer IV, and (2) laminar differences in physiological susceptibility to monocular deprivation. As originally described by Rakic [57], it is now clear that ocular dominance columns in layer IV are already present in a state of incomplete segregation at birth or prior to the onset of the critical period in a number of species [10,11,30]. This early development of incompletely segregated columns is a process independent of visual input, possibly guided by molecular cues. Over the next several weeks, a different process guides the formation of mature ocular dominance columns in layer IV. This process is dependent on visually driven neuronal activity since it does not occur in dark reared animals, where layer IV ocular dominance columns remain in a state of incomplete segregation [51,62]. It is during this period that monocular deprivation can alter the relative size of eye specific columns. In older animals, ocular dominance columns are immutable. In cats, the process leading to incompletely segregated columns is complete by 2 weeks [10], the process of activity dependent ocular dominance segregation is completed at about 6 weeks [39] and ocular dominance columns are immutable by 12 weeks in cats and monkeys [31,51]. The available data do not show a close association between relative NMDA receptor subunit expression (hence subunit composition) and layer IV ocular dominance column formation and plasticity. During the early postnatal process of partial segregation of columns, all three subunits are expressed at very low levels in layer IV, but at high levels in layers that do not receive geniculo-cortical afferents. During the phase of ocular dominance column sharpening and plasticity, only NR1 is expressed at relatively high levels in layer IV and this subunit could play a role in ocular dominance column sharpening and plasticity as suggested previously [7]. Expression of NR2A and NR2B reaches high levels in layer IV only in older cats (after 10 weeks), when ocular dominance

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columns are immutable. It is unlikely, therefore, that NR2A and NR2B subunits play a major role in the process of ocular dominance column formation or plasticity directly at the geniculo-cortical synapse (see below). The present results relate more clearly to laminar differences in susceptibility to the physiological effects of monocular deprivation. The physiological effects of monocular deprivation were classically thought to result from anatomical reorganization of thalamocortical input to ocular dominance columns in layer IV [1,32,40,60]. However, studies of susceptibility to monocular deprivation in dark reared cats [50] and of residual plasticity in visual cortex of more mature normally reared cats [16] indicated dramatic ocular dominance changes in the extragranular layers without significant alterations in layer IV. Moreover, recent evidence indicated that the initial effect of monocular deprivation occurs in cells located in extragranular layers, prior to physiological effects within layer IV [64]. Thus, there is an extragranular mechanism for critical period plasticity that is the primary factor in neonatal monocular deprivation, persists longer in the normal maturation of visual cortex, and is selectively maintained by dark rearing. An intriguing implication of these results is that there is complex retrograde signaling from the extragranular layers back to cells in layer IV, which then feed back to geniculo-cortical afferents to guide relative expansion and contraction of ocular dominance patches in layer IV. The present results indicate a close association of NMDA subunit expression with extragranular layers, particularly for NR2A and NR2B. From 1 week postnatal to near the peak of the critical period, all three NMDA receptor subunits are highly concentrated in extragranular layers and relatively avoid layer IV. NR2A and NR2B subunits continue to be selectively elevated in extragranular layers until beyond 10 weeks, that is, throughout the period when physiological critical period plasticity is maximal. In older animals, there is expression of all three subunits throughout the cortical laminae, but expression remains highest in superficial layers I, II and III for each subunit. This expression pattern in older animals may be related to residual plasticity in adult visual cortex, which is largely restricted to these superficial layers [14].

Acknowledgements This work was supported by National Science Foundation (NSF) Grant 0090777 and NSF EPSCoR Grant EPS-9874764.

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