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The Journal of Neurophysiology Vol. 80 No. 3 September 1998, pp. 1021-1032
Copyright ©1998 by the American Physiological Society
Department of Anatomy, Visual/Motor Neuroscience Division, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0709
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Roberts, Elizabeth B., M. Alex Meredith, and Ary S. Ramoa. Suppression of NMDA receptor function using antisense DNA blocks ocular dominance plasticity while preserving visual responses. J. Neurophysiol. 80: 1021-1032, 1998. Pioneering work has shown that pharmacological blockade of the N-methyl-D-aspartate (NMDA) receptor channel reduces ocular dominance plasticity. However, the results also show that doses of NMDA receptor antagonists that have an effect on ocular dominance plasticity profoundly reduce sensory responses and disrupt stimulus selectivity of cortical cells. It is, therefore, not possible to determine whether effects of NMDA receptor blockade on visual plasticity result from a specific role of NMDA receptors or from the reduction in sensory response. We have used an alternate approach to examine this question. We performed knockdown experiments using antisense oligodeoxynucleotides (ODNs) complementary to mRNA coding the NR1 subunit of the NMDA receptor. After 5 days of antisense, but not sense, ODN treatment NMDA receptor-mediated synaptic transmission was reduced markedly relative to the 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor response, as indicated by whole cell patch-clamp recordings in the cortical slice preparation. This suppression of NMDA receptor-mediated currents was due to a selective reduction in the NR1 protein near the injection site relative to the untreated hemisphere in the same animal, as indicated by immunocytochemistry and Western blotting. In contrast, AMPA receptors were not affected by the antisense ODN treatment indicating specificity of effects. Another major effect of this treatment was to decrease ocular dominance plasticity. Ferrets that were monocularly deprived 1 wk during the antisense ODN treatment had ocular dominance histograms similar to those found in untreated, nondeprived animals. In contrast, ferrets treated with sense ODN and monocularly deprived had ocular dominance histograms resembling those of untreated, monocularly deprived animals. The effects on ocular dominance plasticity did not result from a disruption of sensory responses because maximum responses as well as orientation and direction selectivity of cortical cells were not affected by the treatment. In conclusion, the present results show that antisense techniques can accomplish more selective manipulations of cortical function than is possible with traditional pharmacological agents. Use of this approach also provides unambiguous evidence for a specific role of NMDA receptors in visual plasticity.
Degradation of the visual input to one eye during a critical period of development leads to amblyopia, a major cause of visual disability. In this condition, connections relaying information from the deprived eye to the visual cortex withdraw while connections relaying information from the experienced eye expand (Hubel et al. 1977 This study is based on a total of 33 cortical cells that were examined using whole cell patch-clamp recording in the in vitro slice preparation and 287 cortical cells that were examined by extracellular recordings conducted in vivo. A total of 12 ferrets were used in the visual physiology experiments: 4 untreated (2 were monocularly deprived and 2 had normal visual input), 3 treated with sense ODN (all were monocularly deprived), and 5 animals treated with antisense ODN (3 were treated for 2 wk with monocular deprivation during the 2nd week and 2 were treated for 5-6 days and had normal visual input). Additionally, immunocytochemistry (n = 4 animals) and Western blotting (n = 7 animals) were conducted to examine the effects of antisense ODN treatment on protein expression. All procedures described in this paper were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.
Antisense ODN application
The ODN sequences used here were 5' CAGCAGGTGCATGGTGCT (antisense) and 5' AGCACCATGCACCTGCTG (sense) (Oligos, Etc., Wilsonville, OR). The sequences were chosen to target the 5' coding region of the NR1 subunit mRNA, which is highly conserved in mammals (>99%), and have been used successfully by other researchers (Wahlestadt et al. 1993). To increase stability, phosphorothioate bonds were incorporated between three terminal nucleotides at the 5' and 3' ends. The ODNs were dissolved in phosphate-buffered saline (PBS, 0.9% NaCl in 0.1 M phosphate buffer) to a concentration of 7 µg/µl. Fluorescent latex microspheres (Lumafluor, Naples, FL; 1 µl) were added to the solution for subsequent identification of the injection site. Infusion of ODNs (0.5 µl/h in every case) was accomplished using osmotic minipumps (Alza 2002 and Alza 1007D for 12- and 5-day infusions, respectively) fitted with a catheter and cannula (28 gauge, beveled, stainless steel).
Intracellular recordings
Animals were killed with an overdose of pentobarbital (100 mg/kg). Slices of untreated and ODN-treated cortices were prepared as described previously (Blanton et al. 1989 Immunocytochemistry
Ferrets were deeply anesthetized with pentobarbital (100 mg/kg) and perfused transcardially with cold 0.9% saline (pH 7.2) followed by cold 4% paraformaldehyde in 0.1 M PBS (pH 7.2). The brains were removed and postfixed in 4% paraformaldehyde, 0.1 M PBS for 4 h at 4°C. The caudal portion of the brain containing the primary visual cortex was vibratome sectioned (thickness, 30 µm) in the coronal plane. Free-floating tissue sections were incubated in 3% hydrogen peroxide for 20 min then washed with 0.01 M PBS. (The remainder of the reaction was performed with 0.01 M PBS.) Sections were incubated in 10% normal horse serum, 2% bovine serum albumin (BSA), PBS for 1 h at room temperature as a blocking step. This was followed by incubation in a solution of primary antibody diluted (1:500) in 2% BSA/PBS for 48-72 h at 4°C on a rotary shaker. The primary antibody was either an anti-NMDAR1 monoclonal mouse IgG (clone 54.1, Pharmingen, San Diego, CA) or an antiglutamate receptor 2 and 4 monoclonal mouse IgG (clone 3A11, Pharmingen) to detect AMPA-type glutamate receptors. A washing step of four washes for 10 min each was performed with 2% BSA/PBS. Sections then were incubated with a horse anti-mouse biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted (1:500) in 2% BSA/PBS for 1 h at room temperature. Washes were performed as above. Sections were incubated for 1 h in the Vectastain Elite ABC complex (Vector Laboratories). Washes were performed again as above. Antibody binding was detected by incubating the sections in a solution of 0.06% cobalt enhanced 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in PBS for 1-3 min. Another set of washes was performed as above. Sections were mounted on chrome-alum subbed slides, dehydrated through graded alcohols (50, 70, 95, and 100% ethanol) and clearing agent, and mounted in Permount. Sections of treated and untreated tissue from the same animals always were processed and analyzed together.
Western blotting
Ferrets were deeply anesthetized with intraperitoneal pentobarbital (100 mg/kg) and killed by decapitation. Primary visual cortex around the minipump infusion site (as visualized by location of the latex microspheres) was dissected out and quickly placed in ice cold dissection buffer (50 mM tris(hydroxymethyl)aminomethane (Tris)-acetate, 10% sucrose, 5 mM EDTA, pH 7.4) containing a freshly added protease inhibitor cocktail of 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine, and 20 µg/ml iodoacetamide. The tissue was placed in a tissue homogenizer with 500 µl of dissection buffer and homogenized by hand until the solution was uniform. A small sample was removed to be used for a protein assay. The remaining homogenate then was diluted 1:1 with Laemmli sample buffer (Bio-Rad, Hercules, CA) and boiled for 10 min in a water bath. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad). Protein samples were separated by size using gel electrophoresis and 10% polyacrylamide minigels (Bio-Rad; 5 µg of protein per lane) and transferred to nitrocellulose. After blocking the membrane overnight with 5% dry milk, 1% normal goat serum in 0.1% Tween-20 [polyoxyethylene sorbitan monolaurate (Biorad Laboratories, Hercules, CA)], 20 mM Tris-buffered saline (TTBS) the membrane was incubated for 1 h at room temperature with the primary antibody diluted (1:1,000) in TTBS. Two primary antibodies were used: rabbit anti-NMDAR1 and rabbit anti-glutamate receptor 2 and 3 (to detect AMPA receptors). Both antibodies were affinity purified polyclonal antibodies (Chemicon, Temecula, CA) used at a dilution of 1:1,000. A series of washes was performed using TTBS (4 washes, 15 min each). Incubation with the secondary antibody diluted (1:1,000) in TTBS was performed for 1 h at room temperature. The secondary antibody was a goat anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma). Another series of washes (as above) was performed followed by ECL detection (Amersham, Buckinghamshire, UK) to visualize the antibody reaction. Band densities were measured by densitometry with an image analysis unit (Imaging Research, St. Catherine's, Ontario, Canada) using the MCID/M4 image analysis software. Pixel intensities were converted to optical densities by using a density wedge. A ratio of optical density from each antisense and sense ODN treated sample was determined by dividing the density measurement of the treated by the untreated cortex sample from the same animal. This served to normalize the data so that densities from different immunoblots could be compared. The means of these ratios (relative optical densities) were analyzed with a Student's t-test to determine significance and graphed as bar charts.
Extracellular recordings in vivo
The animals were anesthetized with intraperitoneal pentobarbital (35 mg/kg) and appropriate levels of anesthesia were ascertained by the loss of withdrawal and cornea-blink reflexes. After tracheal cannulation, anesthesia was maintained at surgical levels by using sodium thiopental (10-30 mg/kg ip), and the animal was paralyzed with pancuronium bromide (0.4 mg/kg). Supplemental doses of pentobarbital and pancuronium bromide were given along with subcutaneous injections of 10% dextrose, 0.9% saline every 2 h throughout the experiment or when heart rate or expired CO2 increased. Body temperature and expired CO2 were monitored continuously. The eyelids were opened, nictitating membranes retracted with pseudoephedrine, the pupils dilated with 1% atropine sulfate, and contact lenses were placed on the cornea.
The effects of antisense ODN treatment were assessed at several levels. Accordingly, our findings are divided into four subsections that reflect these levels. In the first section, the synaptic effects were examined using whole cell patch-clamp recording techniques in visual cortical slices. We demonstrate that antisense ODN treatment targeting the NR1 subunit selectively reduced NMDA-receptor function. In the second part, effects on protein expression were examined using specific antibodies. We present evidence for a concomitant reduction in NR1 subunit expression that may underlie the changes in synaptic function. Next, in vivo electrophysiological recordings show that NR1 subunit knockdown prevents the effects of monocular visual deprivation on cortical ocular dominance. In the final section, we show that the reduction in plasticity occurs despite preservation of normal visual responsiveness and stimulus selectivity of cortical cells.
Effects on cortical synaptic transmission
Intracortical application of antisense ODN targeting the NR1 subunit induced a selective reduction of NMDA receptor-mediated synaptic transmission in visual cortex. Intracellular recordings of visual cortical neurons conducted at +40 mV revealed excitatory postsynaptic currents (EPSCs) evoked by stimulation of the cortical white matter in normal, antisense ODN-treated and sense ODN-treated cells (Fig. 1A). To estimate the relative contributions of the AMPA and NMDA receptors to the synaptic responses, the NMDA receptor antagonist D-APV (50 µM) was applied to the bathing medium. This reduced the amplitude of the late component of the synaptic response (Fig. 1A). The short-lasting response remaining during application of D-APV was mediated by AMPA receptors because it was blocked by subsequent bath application of the AMPA receptor antagonist NBQX (10 µM; n = 5 cells, not shown). The EPSCs in cortical neurons recorded within 2-5 mm of the injection site of antisense ODN differed markedly from the EPSCs in normal cortical cells and sense ODN-treated cortical cells. In the antisense treated cortex, the NMDA component of the response (Fig. 1A, dark area) was reduced relative to the AMPA component of the response. This is quantified in the histogram of Fig. 1B, which shows that the average ratio of NMDA-receptor/AMPA-receptor components at the peak of the EPSC for each cell was reduced significantly in the antisense ODN-treated group compared with normal and sense ODN-treated animals (P < 0.01; 8 antisense ODN-treated cells, 9 normal cells, 6 sense ODN-treated cells). A gradient of this effect was observed within antisense treated cortex such that effects at 3.5 mm from the injection site were less evident than at 2 mm from the injection site (Fig. 1A). The sense ODN control was obtained at 2 mm distance from the injection site.
Effects on protein expression
To ascertain that the suppression of NMDA receptor-mediated synaptic currents was due to a selective reduction in the NR1 subunit protein, we conducted immunocytochemistry studies using a monoclonal antibody to the NMDAR1 subunit. To reduce the possibility of artifacts from inadvertent differences in processing, sections of treated visual cortex always were processed together with sections obtained from the contralateral, untreated hemisphere of the same animals. The photomicrographs in Fig. 3 illustrate the finding that antisense ODN treatment (Fig. 3, B and D) induced a reduction in NR1 subunit expression within 2-5 mm of the injection site when compared with normal (Fig. 3, A and C) ferret visual cortex. The higher-power photomicrographs indicate that most cells show a reduced staining intensity (compare Fig. 3, A with B), and the lower-power photomicrographs show that effects were present over a large area of cortex (compare Fig. 3, C with D), corresponding to the region where electrophysiological recordings were conducted (see further). Additionally, the immunocytochemical micrographs, as well as adjacent Nissl stained sections (not shown), indicate the normal histological structure that was observed in ODN treated cortex. Specificity of the antisense ODN treatment was determined by examining expression of the AMPA-type glutamate receptor using an anti-AMPA monoclonal antibody. There is no significant difference in the expression of AMPA receptors between normal (Fig. 3E) and antisense treated (Fig. 3F) ferret visual cortex as seen in immunocytochemistry.
Effects on ocular dominance plasticity
Having established that selective reduction of NMDA-receptor-mediated synaptic currents can be obtained with the antisense ODN treatment, we examined the effects of monocular deprivation on the visual responses of cortical cells in treated animals. Treatment started ~P50 and lasted
Effects on maximum visual response and stimulus selectivity
Finding that antisense ODN treatment blocks visual plasticity raises the question of whether the effects result from a disruption of visual responses. Our results show that treatment did not affect visual responsiveness or stimulus selectivity of cortical cells. Examples of visual responses (e.g., peristimulus histograms) and the corresponding orientation tuning function are shown in Fig. 6, A and C, respectively. These were obtained from a highly responsive, orientation-selective cell located in a hemisphere treated 12 days with antisense ODN targeting the NR1 subunit. For comparison, the orientation tuning function of a cortical cell from an untreated hemisphere is shown in Fig. 6B. These results illustrate the finding that antisense ODN treatment did not affect orientation selectivity of cortical cells. To quantify these results, an orientation selectivity index was obtained for each cell by dividing the response at 45° from the optimal (Fig. 7A) or the response obtained 90° from the optimal (Fig. 7B) by the response at the optimal orientation and subtracting the results from one. Indices of 1.0 indicate a high degree of selectivity, and indices of zero indicate lack of selectivity. The orientation selectivity index was not altered in the antisense ODN-treated (n = 30 cells) and sense ODN-treated cortex (n = 33) relative to normal (n = 55; P > 0.05, Wilcoxon rank sum test). To examine whether antisense ODN treatment affected cortical cells' responses during an early part of the period of monocular deprivation, we have conducted in vivo physiology in additional animals. These animals received minipump implantation at P50 and were studied at P55 rather than P62. The pooled results for these animals (Fig. 7, A and B) were indistinguishable from the other groups, indicating a similar lack of effects at 5 and 12 days of antisense ODN treatment.
The present report presents three major findings concerning the function of NMDA receptors in the developing visual cortex. First, knockdown of NR1 subunits reduces NMDA receptor function in visual cortical neurons. Second, reduction of NMDA-receptor function using antisense techniques is advantageous in that it preserves visual responsiveness and stimulus selectivity of striate cortical neurons. Third, knockdown of the NR1 subunit affects ocular dominance plasticity despite minimal effects on visual activity. The notion that the NMDA receptor is involved in visual development is not new (Constantine-Paton et al. 1990 Antisense oligonucleotides as specific tools to suppress NMDA receptor function
Antisense oligonucleotide treatment is a novel approach to studying visual development. We were, therefore, especially concerned about potential problems with this technique (for a discussion, see Wahlestedt 1994 Role of NMDA receptors in ocular dominance plasticity
Neuronal activity is thought to play a critical role in the circuit rearrangements that characterize the developing mammalian brain and the visual cortex in particular (Katz and Shatz 1996 Role of NMDA receptors in visual cortical responses
Our findings raise the issue of why antisense techniques may produce more specific effects on visual cortex than the currently used pharmacological agents. One possibility is that antisense ODN has more specific effects on visual cortex than conventional pharmacology because the ODN hybridizes only a unique complementary mRNA molecule and intervenes specifically in the function of a single protein. In contrast, some of the traditional pharmacological antagonists may have less specific effects. One example is the competitive antagonist MK-801, which has been reported to block nicotinic cholinergic receptors when used at a low concentration (Ramoa et al. 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Wiesel and Hubel 1965). As a consequence, visual function mediated by the deprived eye can be completely and permanently lost. There is no doubt regarding the substantial scientific and clinical relevance of this type of neural plasticity. However, the cellular and molecular mechanisms underlying this crucial developmental process remain largely unknown.
; Fox and Daw 1993). This receptor may detect correlated pre- and postsynaptic activity (Bourne and Nicoll 1993; Fox and Daw 1993) and subsequently facilitate strengthening of the participating synapses (e.g., from the normal eye) while permitting the loss of terminals that exhibit uncorrelated firing (e.g., from the deprived eye). Although pioneering pharmacological studies have indicated the importance of NMDA receptors in visual plasticity (Bear et al. 1990; Daw 1994; Rauschecker et al. 1990), the results also indicated that doses of NMDA receptor antagonists affecting ocular dominance plasticity lead to a profound depression of sensory responses and loss of orientation and direction selectivity of cortical cells (Bear et al. 1990; Daw 1994; Kasamatsu et al. 1998; Miller et al. 1989; Rauschecker et al. 1990). These findings raise some important questions: are sensory responses invariably compromised by reducing NMDA receptor function and can the effects of reduced NMDA receptor function on visual plasticity be dissociated from the reduction in sensory responsiveness? These are crucial issues because it is difficult to determine whether effects of the pharmacological treatment result from a specific role of NMDA receptors in visual plasticity or from the depression of sensory responses. Moreover, according to some models of visual plasticity (Bienenstock et al. 1982), a reduction in the stimulus selectivity of visual cortical cells may be sufficient to cause a loss of plasticity.
; Kutsuwada et al. 1992; Laurie and Seeburg 1994; Meguro et al. 1992; Monyer et al. 1992; Nakanishi et al. 1992). It has been shown that NMDA receptors lacking the NR1 subunit cannot function normally (Ishii et al. 1993; Kutsuwada et al. 1992; Meguro et al. 1992; Monyer et al. 1992; Moryioshi et al. 1991). Infusion of an antisense ODN targeting the NR1 subunit mRNA should decrease the pool of available NR1 subunits (Agrawal 1996). The new NMDA receptors then should be assembled from the remaining NR2 subunits (Brose et al. 1994; Petralia et al. 1994). Our working hypothesis is that antisense ODN infusion will have more specific effects on the visual cortex than traditional pharmacological agents because the infused DNA binds to a unique complementary mRNA molecule, thereby specifically interfering with the functional expression of a single protein. In contrast, some of the traditional pharmacological antagonists of NMDA receptors are known to have nonspecific effects, such as blockade of non-NMDA receptors (Evans et al. 1982; Jones et al. 1984; Ramoa et al. 1990) and may be toxic when applied locally (Rauschecker et al. 1990). Both effects could contribute to the disruption of visual responses.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Ramoa and McCormick 1994). Recordings were obtained in layers II-IV and started ~2 h after the slices were placed in the recording chamber. Slices were maintained at a temperature of 22°C and superfused with buffered and oxygenated solution containing (in mM) 126 NaCl, 2.5 KCl, 2-4 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 1,000-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 1994). Patch electrodes (4-8 M
) were filled with the following solution (in mM): 120 cesium gluconate, 10 NaCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, 1 sodium ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 MgCl2, 0.1 CaCl2, 2 Na-ATP, and 0.1 Na-GTP, maintained at pH 7.25. Whole cell recordings were conducted at +40 mV in the presence of bicuculline methiodide (30 µM). Bath application of D-aminophosphonovalerate (D-APV; 50 µM) and 6-nitro-7sulfamoylbenzo[f]-quinoxaline-2,3-dione (NBQX; 10 µM) was used to reversibly block NMDA and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, respectively. This test revealed the NMDA and AMPA receptor contributions to the synaptic response. In additional cells located in layer IV, recordings were performed in the presence of NBQX (10 µM) and bicuculline methiodide (30 µM). To determine the intensity-response curve of the NMDA-receptor-mediated synaptic response, different intensities of stimulation, from subthreshold level to levels at which saturation of the response occurred, were applied to white matter. Recordings were obtained using an Axopatch-1D amplifier, digitized using a Neuro-corder encoding unit (Neurodata Instruments, New York, NY) and stored on VCR tape for subsequent analysis. The pClamp data acquisition program (Axon Instruments, Foster City, CA) was used to acquire and analyze data off-line.
) was lowered into the primary visual cortex 2-5 mm away from the minipump cannula using a microdrive device. After the isolation of a single unit, its receptive field, ocular dominance group, and preferred orientation, direction, and velocity were determined qualitatively with a moving bar of light. All data were collected from cells with receptive fields in the binocular region of the visual field. Ocular dominance and orientation and direction selectivity were then quantitatively determined for each cell in the following manner. Under computer control, a moving bar of light (0.5° wide and 20° long) was presented to each eye individually at 5-10 orientations centered around the optimal. One stimulus presentation consisted of the bar of light moving across the receptive field in one direction and back across in the opposite direction. Spikes were collected by the computer during 10 stimulus presentations at each orientation and peristimulus histograms were generated. Spontaneous activity was determined by recording activity in the absence of stimulation. To provide a quantitative estimate of response properties, a binocularity index, an orientation selectivity index, and a direction selectivity index were obtained for each cell as described in RESULTS. Both the orientation and direction selectivity indices were determined from responses to stimulus presentation to the dominant eye. To analyze the results statistically, the median of the binocularity indices was used as a "shift index" for each animal. These shift indices were submitted to a one-way analysis of variance in which a significant F value was obtained. A multiple comparison procedure then was performed to determine which groups were significantly different from one another. Direction and orientation selectivity, spontaneous activity, and maximum responses were analyzed using a Wilcoxon rank sum test.
12 h (or in paraformaldehyde for 4 h) at 4°C. The primary visual cortex was vibratome sectioned (50 µm) in the coronal plane and stained with cresyl violet. These sections were used to reconstruct electrode recording tracts and determine effects of antisense ODN injection on cortical histology. Effects consisted of nonspecific damage caused by the needle used for infusion. This damage was restricted to a volume up to 1 mm from the injection site, beyond which the cortex was indistinguishable from normal, untreated cortex.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 1.
Antisense oligodeoxynucleotide (ODN) treatment selectively reduced the N-methyl-D-aspartate (NMDA) receptor component of synaptic responses in visual cortical neurons. A: whole cell patch-clamp recordings in normal, antisense ODN-treated cortex at 2 different distances away from the injection site (2 and 3.5 mm) and sense ODN-treated cortex 2 mm away from the injection site. Recordings were conducted at +40 mV using a cesium-based solution in the presence of the 
-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (30 µM). Bath application of the NMDA-receptor antagonist D-aminophosphonovalerate (D-APV) was used to block reversibly the NMDA receptor, thereby revealing its contribution to the synaptic response. After 5 days of ODN treatment, the NMDA receptor contribution (shaded area) to the excitatory synaptic currents was markedly reduced in the antisense, but not sense, ODN-treated cortex relative to normal. B: histograms show the average ratio of NMDA receptor/-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor components at the peak of the excitatory postsynaptic current (EPSC) for each cell (n = 23) in the different groups.
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FIG. 2.
Maximum NMDA-EPSC amplitude in layer IV cells was reduced by the antisense ODN treatment. Examples of NMDA-EPSCs obtained from 2 cells in untreated and antisense ODN-treated cortex are shown. Amplitude of the NMDA-EPSC in both cells increased in response to increasing amplitude of white matter stimulation. However, the maximum amplitude observed in the antisense ODN-treated cortex was markedly reduced relative to normal (P < 0.01; n = 10 cells). A plot of NMDA-EPSC amplitude vs. the amplitude of white matter stimulation also is illustrated for these 2 cells. Recordings were conducted at +40 mV in the presence of NBQX (10 µM) to block AMPA receptors and bicuculline methiodide (30 µM) to block GABAA receptors.
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FIG. 3.
Widespread, selective effects of antisense ODN treatment on expression of the NR1 subunit of the NMDA receptor. Immunocytochemistry of normal (A and C) and antisense ODN-treated (B and D) ferret cortex using an anti-NR1 antibody. Low power (×40) photomicrographs (C and D) show that the effect of the antisense ODN treatment on NR1 expression extends over a distance of several millimeters. This area corresponds to the region from which extracellular recordings were obtained in antisense ODN treated animals (Bar, 0.5 mm). High power (×200) photomicrographs (A and B) show that most cells have reduced NR1 immunoreactivity after antisense-ODN treatment (Bar, 50 µm). Immunocytochemical staining with an antibody to AMPA receptor proteins, GluR2 and GluR4, shows no difference in the amount of protein expression between normal (E) and antisense ODN-treated (F) cortex. Normal and antisense ODN-treated tissue were processed together. Sections are from opposite hemispheres of the same animal.
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FIG. 4.
Quantification of the effects of antisense ODN vs. sense ODN treatments on NR1 and AMPA protein expression. Lanes in both immunoblots contain the same samples. Lane 1: antisense ODN-treated visual cortex; lane 2: normal visual cortex from antisense-treated animal; lane 3: sense ODN-treated visual cortex; lane 4: normal visual cortex of sense-treated animal. Immunoblots are shown using anti-NR1 subunit (A) and anti-AMPA receptor (B) antibodies. Bar graphs show that there is a significant reduction in the expression of NR1 subunits (A, P < 0.01) but not AMPA receptors (B, P > 0.5) in the antisense ODN-treated cortices relative to sense-treated cortices. Relative optical densities are the ratios of band densities of treated over untreated hemisphere samples. Means ± SD are graphed in the bar charts (n = 7 samples from different hemispheres in NR1 graph, and n = 5 in AMPA graph). Molecular weights corresponding to the bands are similar to those observed in rat visual cortical samples (not shown), indicating that the antibodies show good cross-reactivity with the ferret proteins.
12 days when ferret cortical neurons show mature orientation selectivity and are still susceptible to effects of monocular deprivation (Chapman and Stryker 1993; Chapman et al. 1996). Extracellular recordings were conducted in the binocular region of the visual field, with the electrode located 2-5 mm away from the injection cannula. To quantify ocular dominance of cortical neurons, we calculated a binocularity index using the following equation: EE/(EE + DE), where EE stands for response to stimulation of the experienced eye (left eye in the case of normal ferrets) and DE for deprived eye (right eye for normal ferrets). A binocularity index of 1.0 indicates that a cell is responsive only to the experienced eye, and a binocularity index of 0.0 indicates that a cell is responsive only to the deprived eye. The histograms showing the distribution of cells into five binocularity ranges (Fig. 5) were compiled from: 48 cells from two untreated ferrets with normal binocular experience (A), 40 cells from two untreated ferrets with 7 days of monocular deprivation (B), 84 cells from three antisense ODN-treated ferrets with 7 days of monocular deprivation (C), and 72 cells from three sense ODN-treated ferrets with 7 days of monocular deprivation (D). Ferrets that were treated with antisense ODN and monocularly deprived during the last week of treatment had ocular dominance histograms similar to normal animals (P > 0.05, Fig. 5, A and C). The expected ocular dominance shift was reduced markedly in these treated animals. In contrast, cortical cells in ferrets that were untreated (Fig. 5B) or treated with sense ODN (Fig. 5D) and monocularly deprived were predominantly driven by the experienced eye, showing ocular dominance histograms that differed significantly from those present in normal animals (P < 0.05).
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FIG. 5.
Knockdown of NR1 subunits prevents the effects of monocular deprivation on ocular dominance profiles. Shift of ocular dominance from the normal profile (A) toward a predominance of the open eye that results from monocular deprivation (B) is prevented by the antisense ODN treatment (C; P < 0.05) but not by the sense ODN treatment (D; P > 0.05), indicating specificity of effects.
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FIG. 6.
Antisense ODN treatment preserves normal stimulus selectivity of individual cortical cells. A: normal response properties in a cortical cell treated for 12 days with continuous intracortical infusion of antisense oligonucleotides to the NR1 subunit. This cell was located ~3 mm from the injection site and was highly selective to the orientation of a moving bar of light as revealed by its orientation tuning function (C). Compare this to the orientation tuning function of a normal, highly selective visual cortical cell in B.
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FIG. 7.
Antisense ODN treatment does not affect orientation or direction selectivity histogram distributions. An orientation selectivity index histogram was created for each treatment group from the responses observed at 45 and 90° from the optimal. Results were pooled together in histograms A and B, respectively. Orientation selectivity indices were not significantly altered in the antisense ODN-treated (5- or 12-day treatments) or sense ODN-treated cortices relative to those of normal ferrets (P > 0.05 in each case, Wilcoxon rank sum test). C: histogram of direction selectivity indices from each cell studied. Direction selectivity also remained unaltered in antisense (5- and 12-day treatments) and sense ODN-treated cortices relative to normal (P > 0.05, Wilcoxon rank sum test).
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FIG. 8.
Antisense ODN treatment preserves maximum visual response of striate cortical cells to a moving bar of light. Responses recorded at the optimal orientation (A) as well as the spontaneous activity (B) of cortical cells were relatively unaffected by treatment. There was no significant difference between any treatment group and normal animals (P > 0.1 for all).
) and that layers II-IV display higher levels of NR1 subunit during the critical period than layers V and VI (Catalano et al. 1997). Stimulus selectivity, maximum response, and spontaneous activity were not differentially affected in these two groups of cells (Wilcoxon rank sum test; P > 0.05). In conclusion, quantification of orientation selectivity, direction selectivity, and visual responsiveness for each cell indicated that antisense ODN treatment did not affect these response properties when compared with those of normal cortical cells and sense ODN-treated cells.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
), and the available experimental evidence suggests the involvement of this receptor in visual plasticity (Bear et al. 1990; Daw 1994; Rauschecker et al. 1990). However, the previous results using pharmacological antagonists of the NMDA receptor were confounded by a disruption of sensory responses that by itself would be sufficient to cause loss of plasticity. In the present study, evidence is presented that the effect of NMDA receptor blockade on visual plasticity can be dissociated from depression of sensory responses and loss of stimulus selectivity of cortical cells. Thus these results provide the first unambiguous evidence for a specific role of NMDA receptors in visual plasticity.
). One possibility is that antisense oligonucleotides may induce nonspecific effects such as downregulation of unrelated receptor proteins. However, several lines of evidence indicate that this possibility is highly unlikely. First, according to available databases, the selected gene region does not share homology with other genes. Second, a previous study of the effects of antisense ODN targeting the NR1 subunit of the NMDA receptor revealed that treatment selectively reduced the density of cortical binding sites of NMDA receptor ligands while preserving the density of binding sites for peptide YY and 6-cyano-7-nitroquinoxaline-2,3-dione (Wahlestedt et al. 1993). Finally, our own series of tests also have indicated the specificity of effects of antisense ODN treatment in the visual cortex. Using the whole cell patch-clamp technique, the relative contributions of the AMPA and NMDA receptors to the synaptic responses were assessed in antisense ODN-treated, sense ODN-treated and normal cortex. Results show that antisense, but not sense, ODN treatment selectively reduced the NMDA receptor contribution to the synaptic response. Similarly, immunocytochemistry and Western blotting revealed that treatment reduced the levels of NR1 subunits near the injection site relative to untreated and sense ODN-treated cortex while leaving AMPA receptors unaffected.
), are ideal for these studies. According to our findings, underivatized antisense failed to prevent the ocular dominance shift (not shown), whereas the analogues used here induced specific physiological effects 2-5 mm away from the injection site. This is a major advantage because previous studies using osmotic minipumps to apply pharmacological agents have indicated that nonspecific damage is evident in a region with a 1 mm radius from the injection site (Ramoa et al. 1988). Histology revealed that damage in animals treated with antisense ODN was restricted to a volume ranging 0.5-1 mm from the injection site, beyond which the cortex was indistinguishable from normal, untreated cortex. Additional evidence for lack of toxicity is the finding of normal visual response properties in cortical cells undergoing ODN treatment. This was true for every cortical layer studied. Collectively, these results indicate that antisense techniques can be used to accomplish more selective manipulations of cortical function than may be possible with traditional pharmacological agents, creating a new and exciting opportunity to examine the molecular mechanisms of visual plasticity.
). The prevailing theory to explain activity-dependent plasticity suggests that mechanisms exist to strengthen synapses the activity of which coincides with target depolarization beyond some threshold level (Hebb 1949) and to eliminate synapses the activity of which is not correlated with postsynaptic activation (Stent 1973). This model requires a correlation detector that would signal synchronous pre- and postsynaptic depolarization (Bourne and Nicoll 1993; Fox and Daw 1993). The biophysical properties of the NMDA receptor suggest that it may function as a correlation detector, playing a critical role in activity-dependent increase in synaptic strength and in synapse stabilization (Bear et al. 1987; Bourne and Nicoll 1993). The ionic channel linked to the NMDA receptor is blocked by Mg2+ at the resting membrane potential (Mayer et al. 1984; Nowak et al. 1984). When the postsynaptic membrane is depolarized sufficiently by strong synaptic stimulation, such as that which results from the synchronous activity of several presynaptic terminals (Bourne and Nicoll 1993; Fox and Daw 1993), Mg2+ blockade of NMDA receptor channels is relieved. Calcium ions then enter the cell via the ionic channel associated with the NMDA receptor (MacDermott et al. 1986). When this influx of calcium exceeds a threshold level, intracellular effector mechanisms would conceivably be activated that lead to synaptic changes (Bear et al. 1987).
). Even the more selective competitive antagonist APV may affect other receptor subtypes when used at a high concentration (Evans et al. 1982; Jones et al. 1984). For instance, intracortical, microiontophoretic application of APV sufficient to block the NMDA receptor response completely also reduced responses mediated by non-NMDA receptors (Fox et al. 1990).
has revealed that APV infusion in the visual cortex caused drastic changes in the physiology as well as histology of cortical neurons over a distance of several millimeters. Finally, another possibility is suggested by the finding that NMDA receptor-mediated synaptic transmission is reduced, but not abolished, by the antisense treatment. This residual function may be important in trophic interactions required for normal cell growth and differentiation. Moreover, the residual function may contribute to sustain the normal sensory responses of cortical cells, which are thought to be partly mediated by NMDA receptors (Fox et al. 1990).
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ACKNOWLEDGEMENTS |
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We thank Dr. Nigel Daw for comments on an earlier version of the manuscript.
This work was supported by grants from the National Eye Institute (1R01 EY-11508-01A2), National Science Foundation (IBN9421983), and the Whitehall Foundation to A. S. Ramoa, and National Science Foundation (IBN9308576) to M. A. Meredith.
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FOOTNOTES |
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Address for reprint requests: A. S. Ramoa, Dept. of Anatomy, Box 98-0709, Visual/Motor Neuroscience Division, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0709.
Received 10 July 1997; accepted in final form 9 April 1998.
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Abstract
Introduction
Methods
Results
Discussion
References
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-phosphonic -carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations.
Br. J. Pharmacol.
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