Relationship of Altered Glutamate Receptor Subunit mRNA Expression to Acute Cell Loss after Spinal Cord Contusion

Experimental Neurology 168, 283–289 (2001) doi:10.1006/exnr.2001.7629, available online at http://www.idealibrary.com on

Relationship of Altered Glutamate Receptor Subunit mRNA Expression to Acute Cell Loss after Spinal Cord Contusion S. D. Grossman, L. J. Rosenberg, and J. R. Wrathall Department of Cell Biology and Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20007 Received April 11, 2000; accepted December 19, 2000

Alterations in the expression of ionotropic glutamate receptors (GluR) contribute to neuronal loss after brain ischemia and epilepsy. In order to determine whether altered expression of GluR subunits might contribute to cell loss after spinal cord injury (SCI), we performed a time course study of subunit mRNA expression using quantitative in situ hybridization. Expression was studied in ventral horn motor neurons (VMN) and glia in adjacent ventral white matter at 15 min and 4, 8, and 24 h after SCI in tissue sections 4 mm rostral and caudal to the injury epicenter. We found that the AMPA subunit GluR2 was significantly downregulated in VMN at 24 h, but not at the earlier times examined, although half the loss of VMN in these locations occurs by 8 h after injury. No changes in the normal expression of GluR2 or GluR4 were found in white matter where glial loss occurs after SCI. NMDA subunits NR1 and NR2A were significantly and rapidly up-regulated in VMN after SCI, but only caudal to the lesion site, while VMN loss is similar rostral and caudal to the epicenter. Thus, the temporal pattern of AMPA and the spatial pattern of NMDA subunit expression changes were distinct from the pattern of VMN loss after SCI. We conclude that altered GluR subunit expression after SCI is unlikely to be involved in secondary cell loss and instead may be involved with plasticity and reorganization of the injured spinal cord. © 2001 Academic Press Key Words: in situ hybridization; glutamate receptors; cell death; spinal cord injury; NMDA; AMPA.

INTRODUCTION

Spinal cord injury (SCI) causes an elevation in extracellular glutamate (28, 39) that activates ionotropic and metabotropic receptors. Ionotropic glutamate receptors (GluRs) are important since they are directly coupled to ion channels and can mediate tremendous alterations in ion concentrations in pathological situations, causing excitotoxic cell death (32). The GluRs are divided into three classes named for their selective

pharmacological agonists: NMDA, AMPA, and kainate (23, 37). Different combinations of subunits confer distinct biochemical and physiological properties to the assembled receptors (3, 4, 20, 25, 33, 43, 55). NMDA receptors comprise at least one NR1 subunit and various combinations of NR2 subunits, named NR2A, B, C, and D (33). AMPA receptors also assemble differentially from a pool of subunits named GluR1, 2, 3, and 4, with the presence of GluR2 subunit conferring calcium impermeability to the receptor (6). Changes in NMDA and AMPA receptor subunits have been repeatedly associated with cell death following brain ischemia (2, 14, 41), amygdaloid kindling (44), and epileptic seizures (11, 30). In particular, decreased AMPA subunit GluR2 expression after ischemia has given rise to the “GluR2 hypothesis,” implicating this alteration and the consequent calcium influx through AMPA receptors devoid of GluR2 as a harbinger of cell death (2, 40). Changes in NMDA receptor subunits have also been seen in cells that eventually die after ischemia (19). Expression of NR1 and NR2C increases (42) in cortex and hippocampus after a hypoxic insult and these changes precede cell death. We have reported (16, 17) specific AMPA and NMDA subunit mRNA changes by 24 h after SCI in ventral horn motor neurons (VMN) after SCI, as well as evidence of altered subunit protein expression in spinal cord homogenates. Secondary loss of VMN rostral and caudal to the injury site appears to be largely accomplished by 24 h after SCI (17) with the extent of loss dependent upon distance from the injury epicenter (59). At 4 mm rostral and caudal to epicenter there was no loss of VMN at 15 min, a significant 30% loss of VMN at 8 h, and increased loss totaling 60% of VMN by 24 h after SCI (66). There was also a significant 20 – 25% loss of glia in the adjacent ventral white matter. We hypothesized that if alterations in GluR subunit expression are directly related to neuronal and/or glial loss after SCI, the mRNA changes should precede cell loss at specific locations. We therefore used in situ hybridization to compare expression of GluR2, GluR4, NR1, and NR2A in VMN and ventral white matter glia

283

0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

284

GROSSMAN, ROSENBERG, AND WRATHALL

at 4 mm rostral and caudal to a standardized contusive SCI. The pattern and extent of alterations of mRNA expression at 15 min and 4, 8, and 24 h after SCI were compared to the temporal and spatial pattern of cell loss at these locations. We confirmed that AMPA and NMDA subunit mRNA expression changes acutely in the first 24 h after SCI, but found that the time course and pattern of changes do not implicate these alterations in cell death. MATERIALS AND METHODS

Spinal cord injury and tissue preparation. SCI was performed as previously described (65). Rats were anesthetized with chloral hydrate (360 mg/kg ip), and a laminectomy was performed at T8. The impounder tip (2.4 mm in diameter) of a weight drop device was lowered onto the dura and a 10 g weight was dropped from a height of 2.5 cm onto the impounder to produce an incomplete spinal contusion. This model has been well characterized (13, 34, 35, 38, 47). Controls were subjected to laminectomy without subsequent SCI. At the specified times after injury, spinal cords were rapidly removed from anesthetized rats (n ⫽ 5 at each time point) and 1.5-mm segments of thoracic cord were frozen and embedded in OCT (Tissue-Tek, Torrance, CA) in sets consisting of segments from injured animals and uninjured controls. Slides representative of each millimeter length of cord were stained with eriochrome RC (46), counterstained with hematoxylin and eosin, and used to reconstruct the injury site, as previously described (60). These sections were also used for VMN and ventral white matter glial cell counts as described in the companion paper (66). Adjacent sections from 4 mm rostral and caudal to the center of the injury epicenter were used in the current study for analysis of GluR subunit mRNA by in situ hybridization. In situ hybridization. Antisense oligonucleotide probes made at the Georgetown University Lombardi Core Sequencing Facility had the following sequences: GluR1 probe sequence is 5⬘ GTC ATC GGT TGT CTG GTC TCG TCC CTC TTC AAA CTC TTC GCT GTG AA 3⬘. The GluR2 probe sequence is TTC ACT ACT TTG TGT TTC TCT TCC ATC TTC AAA TTC CTC AGT GTG. GluR3 sequence is AGG GCT TTG TGG GTC ACG AGG TTC TTC ATT GTT GTC TTC CAA GTG. GluR4 is CTG GTC ACT GGG TCC TTC CTT CCC ATC CTC AGG TTC TTC TGT GTG (24). NR1 is TTC CTC CTC CTC CTC ACT GTT CAC CTT GAA TCG GCC AAA GGG ACT, NR2A is AGA AGG CCC GTG GGA GCT TTC CCT TTG GCT AAG TTT C, NR2B is GGG CCT CCT GGC TCT CTG CCA TCG GCT AGG CAC CTG TTG TAA CCC (4, 24). Oligonucleotides were end-labeled (kit from Boehringer Mannheim, Indianapolis, IN) with [ 35S]dATP (NEN, Boston, MA; sp act 12.5mCi/ml) to yield labeled probes with specific

activities that ranged from 500,000 to 1,000,000 cpm per 0.05 pmol DNA. For in situ hybridization, tissue sections were postfixed, acetylated, and dehydrated as previously described (16). Hybridization solution was placed on each slide (100 ␮l) and incubated (37– 40°C for 20 –24 h) in a petri dish lined with wet filter paper. After a series of washes, slides were air-dried and dipped in Kodak (Rochester, NY) NT2B emulsion, sealed in light-tight boxes, and kept at 4°C for 5– 6 weeks. Slides were developed with D19 developer, fixed with general-purpose fixer (Kodak), and counterstained with neutral red for visualization of cell bodies. Grain counting was done using a computerized image analysis system (Scion Image, Frederick, MD), which quantified the area of the cell body (␮m 2) and counted the overlying grains. These values were used to calculate the average density of grains (grains/␮m 2) for a given VMN. In the case of white matter, grains were counted in a 0.2-␮m 2 area bordered by the ventromedial sulcus and the ventral pial surface. All values were corrected for background by sampling adjacent areas of the slide devoid of tissue and subtracting the density of grains in the background from that in experimental samples. Statistical significance was determined using SigmaStat (SPSS, San Raphael, CA). The averages for each rat were used to calculate means and standard errors for each time point we examined. Analysis of variance (ANOVA) followed by a post hoc test (Tukey) was performed with P values set at 0.05 to establish statistical differences between specific groups or time points. RESULTS

In situ hybridization results using antisense oligonucleotide probes for AMPA subunits GluR1, GluR2, GluR3, and GluR4 and NMDA subunits NR1, NR2A, and NR2B from control spinal cord sections were consistent with what we have previously published (16, 17), with each probe labeling a particular pattern of cells in the spinal gray matter. While there was no white matter labeling for NMDA subunits, we did see white matter labeling for GluR2 and GluR4 that was above background (Fig. 1). We quantified GluR2 mRNA at 15 min and 4, 8, and 24 h after SCI in VMN and in ventromedial white matter 4 mm distal to the lesion epicenter. Grains overlying large VMN counterstained with neutral red were counted. We found no evidence of a difference in GluR2 mRNA in VMN at 15 min after injury compared to controls (Fig. 2). Analysis at 4 h also showed that GluR2 mRNA was still within control levels in physically intact VMN. At 8 h, when there was a significant 30% VMN loss (66), no change in GluR2 mRNA was detected. By 24 h, GluR2 mRNA in VMN was significantly down-regulated to approximately 60% of control

GluR SUBUNITS IN CELL LOSS

285

FIG. 3. Frequency histograms of GluR2 grain distributions in VMN. (A) At 8 h after SCI the frequency histogram is coincident for VMN in SCI and laminectomy control tissue. (B) At 24 h after SCI the frequency histogram is still monomodal, but the curve is shifted to the left with the average grain density reduced compared to controls. Squares indicate control VMN expression of GluR2 as assessed by counting number of grains overlying the neuronal cell bodies. Triangles represent data from VMN analyzed from SCI rats. Data points represent means ⫾ standard errors. FIG. 1. Normal expression of NR1, GluR2, and GluR4 mRNAs in ventral motor neurons and ventromedial white matter. In these bright-field micrographs, the small black grains overlying cells represent GluR subunit mRNA expression. Normal expression levels of NR1 mRNA are high in VMN (A) but not detectable in ventromedial white matter (D) as viewed by bright-field microscopy. GluR2 mRNA is also high in VMN (B) and moderately expressed in ventromedial white matter (E). GluR4 mRNA is lower in VMN (C) but also moderately expressed in ventromedial white matter (F). Scale bar, 0.05 ␮m.

levels, consistent with what we have previously reported (16). At 24 h about 60% of VMN at ⫾4 mm are lost (66) and the VMN loss appears similar at 24 h and 1 month after SCI (17). Thus, half of the VMN expected to die at this location were already lost before detection of decreased GluR2 mRNA. The possibility remained that a subpopulation of VMN that expressed relatively lower amounts of GluR2 mRNA was selectively lost. In order to address this issue, we constructed frequency histograms at 8 and 24 h to examine the distribution of GluR2 grains among VMN. At 8 h virtually identical frequency his-

FIG. 2. AMPA subunit GluR2 is down-regulated at 24 h in VMN surrounding the lesion epicenter after SCI. Grain counts were done for GluR2 mRNA in VMN 4 mm rostral and caudal to the lesion epicenter. *Statistical significance by ANOVA with post hoc Tukey, P ⬍ 0.05.

tograms were seen for VMN in SCI and control tissue (Fig. 3A). In both cases a single population of cells was indicated with no evidence of a bimodal grain distribution that would suggest two subpopulations of VMN with respect to GluR2 mRNA expression. At 24 h after SCI, the frequency distribution remained monomodal but was shifted to the left, indicating that remaining VMN expressed fewer GluR2 grains than their counterparts in uninjured control spinal cords (Fig. 3B). These data indicate there is no subpopulation of VMN with lower GluR2 mRNA expression that might be selectively lost after SCI. We previously detected no difference in AMPA subunits GluR1 and GluR3 at 24 h after injury (16). We examined expression of GluR1 and GluR3 mRNA at 4 h post injury (Fig. 4), but detected no difference in mRNA levels of either subunit at this earlier time point. GluR4, which was down-regulated 20% at 24 h (16), was not decreased at the earlier 4-h time point (Fig. 4). We also examined expression of GluR2 and GluR4 mRNA in ventromedial white matter after SCI since

FIG. 4. AMPA subunit mRNAs are not changed at 4 h after SCI. Graph represents grain counts expressed as percentage of control for AMPA subunits GluR1, GluR2, GluR3, and GluR4 at 4 h after SCI in VMN 4 mm distal from the lesion epicenter. Bars represent means ⫾ SE.

286

GROSSMAN, ROSENBERG, AND WRATHALL

FIG. 5. NMDA subunits NR1 and NR2A mRNAs are up-regulated in VMN below the lesion epicenter after SCI. (A) Grain counts were done for NR1 mRNA in VMN 4 mm caudal to the lesion epicenter. Significant up-regulation is seen at 4, 8, and 24 h after SCI. (B) Grain counts for NR2A mRNA reveal a significant upregulation of NR2A in VMN 4 mm below the lesion epicenter at 15 min and 4, 8, and 24 h after SCI. Bars represent means ⫾ SE. *Statistical significance by ANOVA with post hoc Tukey, P ⱕ 0.05.

these subunits are expressed by glial cells (58). Using the same sections as analyzed for VMN data, a 0.2mm 2 area of ventromedial white matter on either side of the ventral sulcus was examined. We found that GluR4 expression in laminectomy controls (1.91 ⫾ 0.27, net grains per ␮m 2) was not changed at 15 min or 4, 8, or 24 h after SCI ( f ⫽ 0.356, P ⫽ 0.84). We analyzed GluR2 at 24 h after SCI, the time when VMN showed significant down-regulation of this subunit, but found no change in GluR2 mRNA expression in the white matter (t ⫽ ⫺1.04, P ⫽ 0.34). We have previously shown an up-regulation of NR1 and NR2A mRNA in VMN below, but not above, the injury site at 24 h after SCI (17). We therefore examined the expression of these mRNAs at 4 mm rostral and caudal to the injury site at earlier times after SCI. At 15 min after injury, quantitative grain counting caudal to the epicenter revealed that NR1 mRNA showed a tendency toward up-regulation that was not statistically significant. By 4 h, however, NR1 mRNA was significantly up-regulated and this up-regulation was maintained at 8 h. At 24 h, NR1 mRNA was even higher, reaching nearly 200% of control values below the epicenter (Fig. 5A). Interestingly, NR2A mRNA was significantly up-regulated by about 60% as soon as 15 min postinjury (Fig. 5B). This up-regulation appeared to be stable and persisted through 24 h. There were no changes in NR1 or NR2A mRNA in rostral VMN at any time examined, nor were there any changes in NR2B mRNA (data not shown). DISCUSSION

The results from this study argue against a causal relationship between alterations in expression of AMPA or NMDA receptor subunits and the secondary death of VMN or glia in the first 24 h after an incomplete contusive SCI.

We have previously reported that at 24 h and 1 month after SCI there were significant alterations in mRNA and protein levels for several glutamate receptor subunits (16, 17). One of these was the AMPA subunit GluR2, which confers Ca 2⫹ impermeability on receptors in which it is present (6). GluR2 mRNA in thoracic VMN and thoracic spinal cord protein levels of GluR2 are down-regulated by about 50% at 24 h after SCI and remain so at 1 month after injury (16). In the current study, examination of more acute time points revealed that down-regulation of GluR2 did not occur until between 8 and 24 h. At least half of the VMN expected to die in the region investigated were lost (66) prior to detectable change in GluR2 mRNA. Given that additional time would be required for translation and assembly of subunits into functional receptors, our data strongly suggest that the formation of Ca 2⫹-permeable AMPA receptors does not contribute to death of VMN in our model of SCI. Our results are in contrast to studies suggesting that down-regulation of GluR2 is a harbinger of cell death (2, 40). Instead, our results indicate that loss of GluR2, known to result in increased calcium influx (14), may result in increased synaptic plasticity. This is consistent with findings by Jia et al. (22) showing that decreased GluR2 results in increased calcium influx and enhances synaptic plasticity as measured by increased long-term potentiation. Studies by others have also shown that loss of GluR2 is not necessarily a marker of selective neuronal vulnerability (64). Our results are not the first to argue against the GluR2 hypothesis (1, 10). Further, decreased GluR2 in our model of SCI does not preclude cell survival, since GluR2 mRNA and protein are still down-regulated at 1 month after SCI in the surviving VMN (16). Turnover rates of GluR subunits. The time differences between detectable AMPA subunit changes and detectable NMDA subunit changes may be explained by differences in the half-lives of the subunits. Studies on turnover rates of AMPA receptors (29) indicate that each subunit has a half-life of about 20 h, measured by surface biotinylation and pulse chase. Thus, even if VMN are signaled to immediately down-regulate AMPA subunits after SCI, we would not expect a substantial change until after 20 h, consistent with our results. This may be why the “GluR2 hypothesis” (2, 14, 41) is not relevant to our model of SCI, in which much of the VMN loss is accomplished by 24 h, in contrast to the delayed cell loss that continues for weeks after brain ischemia. Modulation of AMPA channels by subunit synthesis is not a rapid and efficient process, and other modes of receptor regulation may be more critical. For example phosphorylation (49, 52, 57) occurs within minutes and causes rapid membrane insertion from an intracellular free pool of subunits

287

GluR SUBUNITS IN CELL LOSS

(26). These other modes of receptor modulation may be active after SCI. In contrast to AMPA subunits, the NMDA subunit NR1 has a much shorter half-life of about 2 h (21, 61), consistent with our findings of rapid changes in NR1 mRNA after SCI. The mRNAs for the NMDA subunits NR2A and NR1 were up-regulated in VMN by 15 min and 4 h, respectively, and continued to increase in the first 24 h in VMN below the lesion site. We did not see these changes in VMN rostral to the lesion site. However, VMN loss is symmetrical, strongly suggesting that changes in NMDA subunits may be associated with the loss of descending innervation but not loss of VMN. NMDA receptors are classically associated with activity-dependent plasticity (63) and synaptic rearrangement (53). Their composition changes in response to sensory experience (7), and subunit expression changes are regulated by neuronal activity (5, 36). NR2A increases during synaptogenesis in correlation with activity-dependent processes during development (56, 61). The rapid increase in NR2A mRNA 15 min after SCI was surprising, but is consistent with findings (9) that NR2A protein is up-regulated in the visual cortex within 1 h of sensory stimulation. Logically, mRNA up-regulation would have to precede this protein upregulation, making it consistent with rapid up-regulation of mRNA as soon as 15 min after SCI. Rapid increases in NR2A protein (45) are blocked by cycloheximide, suggesting that new subunits are being translated and not just moving from one cellular compartment to another. The doubling in NR2A mRNA in VMN below the lesion seen in our model may represent an up-regulation of functional NMDA receptors in these cells that are of the NR1/2A composition. Thus, NR2A production may be under inducible regulation (9) by synaptic activity (5, 36). New NMDA receptors may be formed that have more of the NR2A subunit, are less sensitive to glutamate, and display very fast decay kinetics (8, 9). This may be beneficial in the compromised environment of the injured spinal cord. The increase in NR1 mRNA is first detected at 4 h, remains up through 24 h, but returns to control levels by 1 month (17). NR1 may be up-regulated acutely after injury but becomes unstable and gets degraded when it is not successful in assembling with NR2s into functional receptors (21). This is the case with other ion channel subunits, such as the ␣ subunit of the muscle nicotinic acetylcholine receptor, the Kv1.2 subunit of potassium channels, and the ␣ subunit of the voltage-sensitive sodium channel (31, 51, 54). Each of these subunits is produced in excess of others and gets degraded when it fails to combine with other subunits. Alternatively, NR2 production may be the rate-limiting step in NMDA receptor expression, such that un-

assembled NR1 serves as a reserve waiting for changes in NR2 expression (21). Are changes in GluR expression due to altered activity? NMDA receptor activity is necessary for the insertion of new NR2A into synaptic NMDA receptors (45). Quinlan et al. proposed that sensory experience activates NMDA receptors and regulates the synthesis and expression of NR2A-containing NMDA receptors in the visual cortex (45). This may be applicable to the situation after SCI, in that excess release of glutamate (28, 39) may stimulate glutamate receptors and cause up-regulation of NR2A. However, glutamate is likely to diffuse equally on either side of the injury site, indicating that some additional factor is present to account for the asymmetry of the subunit alteration after SCI. Changes in synaptic activity may also change GluR subunit expression (15, 27, 36, 62). Blocking activity chronically with NMDA antagonists or sodium channel blockers in hippocampal neurons in culture increases NR2A levels and increases NMDA receptor binding sites by over 300% (48). The reverse is also seen. Cerebellar granule cells in culture, when treated with NMDA agonists, down-regulate NR1 mRNA and protein (50). NMDA subunit changes after SCI could be due to the loss of descending activity. Blocking axonal conduction in the spinal cord with a sodium channel blocker and examining GluR changes may be used to elucidate the role of activity on GluR expression. We have recently found (18) that focal injection of tetrodotoxin in the thoracic spinal cord produces hind limb paralysis at 24 h, similar to that seen after SCI, and also increases expression of NR1 and NR2A mRNA in VMN, as occurs at 24 h after SCI. Thus, the up-regulation of NR1 and NR2A mRNA after SCI appears to be “inactivity dependent.” Overall, we find that altered expression of glutamate receptor subunits does not precede cell loss in a manner suggestive of causality. Our results are consistent with findings by Fryer et al. (12), who showed that a difference in glutamate receptor subunit expression is unlikely to predict vulnerability to cell death during normal development, but does affect synaptic plasticity. Although there is tremendous cell loss associated with SCI and significant effects on AMPA and NMDA receptor subunits, our evidence suggests that these two events are not linked. Instead, changes in GluR subunit expression may be involved with reorganization and plasticity of the spinal cord after injury. ACKNOWLEDGMENTS This study was supported by National Institutes of Health Grants RO1 NS 37733 and RO1 NS 35647 (J.R.W.) and NIMH NRSA 1F31 MH 12038 (S.D.G.).

288

GROSSMAN, ROSENBERG, AND WRATHALL

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Ben-Ari, Y., and M. Khrestchatisky. 1998. The GluR2 (GluRB) hypothesis in ischemia: Missing links. Trends Neurosci. 21: 241–242. Bennett, M. V., D. E. Pellegrini-Giampietro, J. A. Gorter, E. Aronica, J. A. Connor, and R. S. Zukin. 1996. The GluR2 hypothesis: Ca(⫹⫹)-permeable AMPA receptors in delayed neurodegeneration. Cold Spring Harbor Symp. Quant. Biol. 61: 373–384. Bochet, P., E. Audinat, B. Lambolez, F. Crepel, J. Rossier, M. Iino, K. Tsuzuki, and S. Ozawa. 1994. Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel. Neuron 12: 383–388. Buller, A. L., H. C. Larson, B. E. Schneider, J. A. Beaton, R. A. Morrisett, and D. T. Monaghan. 1994. The molecular basis of NMDA receptor subtypes: Native receptor diversity is predicted by subunit composition. J. Neurosci. 14: 5471–5484. Buonanno, A., and R. D. Fields. 1999. Gene regulation by patterned electrical activity during neural and skeletal muscle development. Curr. Opin. Neurobiol. 9: 110 –120. Burnashev, N., H. Monyer, P. H. Seeburg, and B. Sakmann. 1992. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron 8: 189 –198. Carmignoto, G., and S. Vicini. 1992. Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 258: 1007–1011. Flint, A. C., U. S. Maisch, J. H. Weishaupt, A. R. Kriegstein, and H. Monyer. 1997. NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J. Neurosci. 17: 2469 –2476. Fox, K., J. Henley, and J. Isaac. 1999. Experience-dependent development of NMDA receptor transmission. Nat. Neurosci. 2: 297–299. Frank, L., N. H. Diemer, F. Kaiser, M. Sheardown, J. S. Rasmussen, and P. Kristensen. 1995. Unchanged balance between levels of mRNA encoding AMPA glutamate receptor subtypes following global cerebral ischemia in the rat. Acta Neurol. Scand. 92: 337–343. Friedman, L. K. 1998. Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss. Hippocampus 8: 511–525. Fryer, H. J., R. J. Knox, S. M. Strittmatter, and R. G. Kalb. 1999. Excitotoxic death of a subset of embryonic rat motor neurons in vitro. J. Neurochem. 72: 500 –513. Gale, K., H. Kerasidis, and J. R. Wrathall. 1985. Spinal cord contusion in the rat: Behavioral analysis of functional neurologic impairment. Exp. Neurol. 88: 123–134. Gorter, J. A., J. J. Petrozzino, E. M. Aronica, D. M. Rosenbaum, T. Opitz, M. V. Bennett, J. A. Connor, and R. S. Zukin. 1997. Global ischemia induces downregulation of GluR2 mRNA and increases AMPA receptor-mediated Ca 2⫹ influx in hippocampal CA1 neurons of gerbil. J. Neurosci. 17: 6179 – 6188. Gottmann, K., A. Mehrle, G. Gisselmann, and H. Hatt. 1997. Presynaptic control of subunit composition of NMDA receptors mediating synaptic plasticity. J. Neurosci. 17: 2766 –2774. Grossman, S., B. Wolfe, R. Yasuda, and J. Wrathall. 1999. Alterations in AMPA receptor subunits following contusive spinal cord injury in the rat. J. Neurosci. 19: 5711–5720. Grossman, S. D., B. B. Wolfe, R. P. Yasuda, and J. R. Wrathall. 2000. Changes in NMDA receptor subunit expression in response to contusive spinal cord injury. J. Neurochem. 75: 174 – 184.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Grossman, S. D., and J. R. Wrathall. 2000. The role of activity blockade on glutamate receptor subunit expression in the spinal cord. Brain Res. 880: 183–186. Heurteaux, C., I. Lauritzen, C. Widmann, and M. Lazdunski. 1994. Glutamate-induced overexpression of NMDA receptor messenger RNAs and protein triggered by activation of AMPA/ kainate receptors in rat hippocampus following forebrain ischemia. Brain Res. 659: 67–74. Hollmann, M., M. Hartley, and S. Heinemann. 1991. Ca 2⫹ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition. Science 252: 851– 853. Huh, K., and R. Wenthold, R. 1999. Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the Nmethyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells. J. Biol. Chem. 274: 151–157. Jia, Z., N. Agopyan, P. Miu, Z. Xiong, J. Henderson, R. Gerlai, F. A. Taverna, A. Velumian, J. MacDonald, P. Carlen, W. Abramow-Newerly, and J. Roder. 1996. Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron 17: 945–956. Kaczmarek, L., M. Kossut, and J. Skangiel-Kramska. 1997. Glutamate receptors in cortical plasticity: Molecular and cellular biology. Physiol. Rev. 77: 217–255. Keinanen, K., W. Wisden, B. Sommer, P. Werner, A. Herb, T. A. Verdoorn, B. Sakmann, and P. H. Seeburg. 1990. A family of AMPA-selective glutamate receptors. Science 249: 556 –560. Krupp, J. J., B. Vissel, S. F. Heinemann, and G. L. Westbrook. 1996. Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol. Pharmacol. 50: 1680 –1688. Liao, D., N. A. Hessler, and R. Malinow. 1995. Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice. Nature 375: 400 – 404. Lissin, D. V., S. N. Gomperts, R. C. Carroll, C. W. Christine, D. Kalman, M. Kitamura, S. Hardy, R. A. Nicoll, R. C. Malenka, and M. von Zastrow. 1998. Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors. Proc. Natl. Acad. Sci. USA 95: 7097–7102. Liu, D., W. Thangnipon, and D. J. McAdoo. 1991. Excitatory amino acids rise to toxic levels upon impact injury to the rat spinal cord. Brain Res. 547: 344 –348. Mammen, A. L., R. L. Huganir, and R. J. O’Brien. 1997. Redistribution and stabilization of cell surface glutamate receptors during synapse formation. J. Neurosci. 17: 7351–7358. Mathern, G. W., J. K. Pretorius, H. I. Kornblum, D. Mendoza, A. Lozada, J. P. Leite, L. M. Chimelli, I. Fried, A. C. Sakamoto, J. A. Assirati, M. F. Levesque, P. D. Adelson, and W. J. Peacock. 1997. Human hippocampal AMPA and NMDA mRNA levels in temporal lobe epilepsy patients. Brain 120: 1937–1959. Merlie, J. P., and J. Lindstrom. 1983. Assembly in vivo of mouse muscle acetylcholine receptor: Identification of an alpha subunit species that may be an assembly intermediate. Cell 34: 747–757. Michaelis, E. K. 1998. Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging. Prog. Neurobiol. 54: 369 – 415. Monyer, H., R. Sprengel, R. Schoepfer, A. Herb, M. Higuchi, H. Lomeli, N. Burnashev, B. Sakmann, and P. H. Seeburg, P. H. 1992. Heteromeric NMDA receptors: Molecular and functional distinction of subtypes. Science 256: 1217–1221. Noble, L. J., and J. R. Wrathall, J. R. 1985. Spinal cord contusion in the rat: Morphometric analyses of alterations in the spinal cord. Exp. Neurol. 88: 135–149. Noble, L. J., and J. R. Wrathall. 1989. Correlative analyses of lesion development and functional status after graded spinal cord contusive injuries in the rat. Exp. Neurol. 103: 34 – 40.

GluR SUBUNITS IN CELL LOSS 36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

Ozaki, M., M. Sasner, R. Yano, H. S. Lu, and A. Buonanno. 1997. Neuregulin-beta induces expression of an NMDA-receptor subunit. Nature 390: 691– 694. Ozawa, S., H. Kamiya, and K. Tsuzuki. 1998. Glutamate receptors in the mammalian central nervous system. Prog. Neurobiol. 54: 581– 618. Panjabi, M. M., and J. R. Wrathall. 1988. Biomechanical analysis of experimental spinal cord injury and functional loss. Spine 13: 1365–1370. Panter, S. S., S. W. Yum, and A. I. Faden. 1990. Alteration in extracellular amino acids after traumatic spinal cord injury. Ann. Neurol. 27: 96 –99. Pellegrini-Giampietro, D. E., J. A. Gorter, M. V. Bennett, and R. S. Zukin. 1997. The GluR2 (GluR-B) hypothesis: Ca(2⫹)permeable AMPA receptors in neurological disorders. Trends Neurosci. 20: 464 – 470. Pellegrini-Giampietro, D. E., R. S. Zukin, M. V. Bennett, S. Cho, and W. A. Pulsinelli. 1992. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. Proc. Natl. Acad. Sci. USA 89: 10499 – 10503. [Published erratum appears in Proc. Natl. Acad. Sci. USA, 1993, 90: 780] Perez-Velazquez, J. L., and L. Zhang. 1994. In vitro hypoxia induces expression of the NR2C subunit of the NMDA receptor in rat cortex and hippocampus. J. Neurochem. 63: 1171–1173. Popovich, P. G., P. Wei, and B. T. Stokes. 1997. Cellular inflammatory response after spinal cord injury in Sprague–Dawley and Lewis rats. J. Comp. Neurol. 377: 443– 464. Prince, H. K., P. J. Conn, C. D. Blackstone, R. L. Huganir, and A. I. Levey. 1995. Down-regulation of AMPA receptor subunit GluR2 in amygdaloid kindling. J. Neurochem. 64: 462– 465. Quinlan, E. M., B. D. Philpot, R. L. Huganir, and M. F. Bear. 1999. Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo. Nat. Neurosci. 2: 352–357. Rabchevsky, A. G., I. Fugaccia, A. Fletcher-Turner, D. A. Blades, M. P. Mattson, and S. W. Scheff. 1999. Basic fibroblast growth factor (bFGF) enhances tissue sparing and functional recovery following moderate spinal cord injury. J. Neurotrauma 16: 817– 830. Raines, A., K. L. Dretchen, K. Marx, and J. R. Wrathall. 1988. Spinal cord contusion in the rat: Somatosensory evoked potentials as a function of graded injury. J. Neurotrauma 5: 151–160. Rao, A., and A. M. Craig. 1997. Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 19: 801– 812. Raymond, L. A., W. G. Tingley, C. D. Blackstone, K. W. Roche, and R. L. Huganir. 1994. Glutamate receptor modulation by protein phosphorylation. J. Physiol. Paris 88: 181–192. Resink, A., M. Villa, D. Benke, H. Hidaka, H. Mohler, and R. Balazs. 1996. Characterization of agonist-induced down-regulation of NMDA receptors in cerebellar granule cell cultures. J. Neurochem. 66: 369 –377. Rhodes, K. J., M. M. Monaghan, N. X. Barrezueta, S. Nawoschik, Z. Bekele-Arcuri, M. F. Matos, K. Nakahira, L. E.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

289

Schechter, and J. S. Trimmer. 1996. Voltage-gated K ⫹ channel beta subunits: Expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain. J. Neurosci. 16: 4846 – 4860. Roche, K. W., W. G. Tingley, and R. L. Huganir. 1994. Glutamate receptor phosphorylation and synaptic plasticity. Curr. Opin. Neurobiol. 4: 383–388. Scheetz, A. J., and M. Constantine-Paton. 1994. Modulation of NMDA receptor function: Implications for vertebrate neural development. FASEB J. 8: 745–752. Schmidt, J. W., and W. A. Catterall. 1986. Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons. Cell 46: 437– 444. Sprengel, R., B. Suchanek, C. Amico, R. Brusa, N. Burnashev, A. Rozov, O. Hvalby, V. Jensen, O. Paulsen, P. Andersen, J. J. Kim, R. F. Thompson, W. Sun, L. C. Webster, S. G. Grant, J. Eilers, A. Konnerth, J. Li, J. O. McNamara, and P. H. Seeburg. 1998. Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92: 279 –289. Stocca, G., and S. Vicini. 1998. Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J. Physiol. (London) 507: 13–24. Swope, S. L., S. J. Moss, C. D. Blackstone, and R. L. Huganir. 1992. Phosphorylation of ligand-gated ion channels: A possible mode of synaptic plasticity. FASEB J. 6: 2514 –2523. Tachibana, M., R. J. Wenthold, H. Morioka, and R. S. Petralia. 1994. Light and electron microscopic immunocytochemical localization of AMPA-selective glutamate receptors in the rat spinal cord. J. Comp. Neurol. 344: 431– 454. Teng, Y. D., I. Mochetti, and J. R. Wrathall. 1997. FGF2 prevents motoneuron death after experimental spinal cord injury. Soc. Neurosci. Abstr. 23. Teng, Y. D., and J. R. Wrathall. 1997. Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury. J. Neurosci. 17: 4359 – 4366. Tovar, K. R., and G. L. Westbrook. 1999. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J. Neurosci. 19: 4180 – 4188. Turrigiano, G. G., K. R. Leslie, N. S. Desai, L. C. Rutherford, and S. B. Nelson. 1998. Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391: 892– 896. Vallano, M. L., B. Lambolez, E. Audinat, and J. Rossier. 1996. Neuronal activity differentially regulates NMDA receptor subunit expression in cerebellar granule cells. J. Neurosci. 16: 631– 639. Vandenberghe, W., W. Robberecht, and J. R. Brorson. 2000. AMPA receptor calcium permeability, GluR2 expression, and selective motoneuron vulnerability. J. Neurosci. 20: 123–132. Wrathall, J. R., R. K. Pettegrew, and F. Harvey. 1985. Spinal cord contusion in the rat: Production of graded, reproducible, injury groups. Exp. Neurol. 88: 108 –122. Grossman, S. D., L. J. Rosenberg, and J. R. Wrathall. 2001. Temporal–spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp. Neurol. 168: 273–282.