- Email: [email protected]
Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices
Neuroscience Letters 232 (1997) 87–90
Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices Daniel L. Small a ,*, Michael O. Poulter a, Alastair M. Buchan b, Paul Morley a a
Institute for Biological Sciences, National Research Council of Canada, Building M-54, Montreal Road, Ottawa, Ontario, K1A 0R6 Canada b Foothills Hospital, 1403-29 Street N.W., Calgary, Alberta, T2N 2T9 Canada Received 4 June 1997; received in revised form 28 July 1997; accepted 29 July 1997
Abstract Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-d-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions. 1997 Elsevier Science Ireland Ltd. Keywords: In vitro ischemia; Excitotoxicity; Reverse transcription-polymerase chain reaction; N-Methyl-d-aspartate receptor; Hippocampus
Given that N-methyl-d-aspartate (NMDA) receptors are important targets of therapeutic intervention [10], and their pharmacological diversity is largely determined by the NR2 subunits of the heteromeric receptor [15], it is relevant to characterize the post-ischemic expression of NR2 subunits to ensure optimal specificity and efficacy of potential neuroprotective compounds. Although several studies have demonstrated changes in NMDA receptor subunit expression following cerebral ischemia [7,8,11,16], there is controversy associated with these changes. Some evidence suggests that there is no change in NR1 expression [7,11, 16], while other evidence suggests that there is a significant increase [8] and in situ hybridization studies suggest there is an increase in NR2A, and NR2B, but not NR2C [8]. In an in vitro hippocampal slice model of cerebral ischemia, PerezVelazquez and Zhang [12] observed a rapid (,45 min) increase in NR2C subunit mRNA expression which, based on preliminary results, also occurred in vivo [12]. Function-
* Corresponding author. Tel.:+1 613 9900902; fax: +1 613 9414475; e-mail: [email protected]
ally, using the in vitro model, Zhang et al. [18] reported a reduced Mg2+ sensitivity of NMDA-mediated currents, a biophysical feature of NR2C-containing receptors [9], in the vulnerable CA1 pyramidal neurons following 18 h of re-oxygenation and return to 4 mM glucose in the hippocampal slice. It is not known whether the increased NR2C expression is restricted to the vulnerable CA1 region of the hippocampus or whether NR2C induction confers resistance or vulnerability. Using reverse transcription-polymerase chain reaction (RT-PCR) amplification we have quantified the relative expression of NR2A-C subunits in rat hippocampal slices exposed to the same oxygen-glucose deprivation insult used by Perez-Velazquez and Zhang [12]. By quantifying the relative NR2 subunit expression in microdissected CA1, CA3 and dentate gyrus (DG) regions of hippocampal slices, we have localized the changes in NR2C expression to resistant and vulnerable regions of the hippocampus. Male Wistar rats (175–200 g) anesthetized with 3% isoflurane were decapitated and their brains were rapidly (,1 min) removed and submerged in cold (4°C), artificial cerebrospinal fluid (ACSF) consisting of (in mM): 127 NaCl, 2
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0592-2
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D.L. Small et al. / Neuroscience Letters 232 (1997) 87–90
KCl, 10 glucose, 1.2 KH2PO4, 26 NaH2CO3, 2 MgSO4, 2 CaCl2 (pH 7.4) bubbled with 95% O2/5% CO2. Transverse hippocampal slices (400 mm) were placed onto floating nylon mesh platforms in covered incubation chambers (750 ml) such that the slices were submerged in 2–3 mm of 95% O2/5% CO2 bubbled ACSF at 34°C. After 90 min, slices that were to be deprived of oxygen and glucose were transferred to smaller chambers containing 30–40 ml of 95% N2/5% CO2 bubbled ACSF with 0 mM glucose for 4 min at 34°C. The temperature, glucose concentration, and duration of insult, were chosen to replicate the insult conditions of Perez-Velazquez and Zhang, [12]. Slices were returned to the 750 ml chamber following the 4 min insult. The CA1, CA3 and DG regions were microdissected in ACSF at 4°C using a 60 × dissecting microscope, fine forceps and a #15 scalpel blade. The first cut was perpendicular to the open, lateral end of the DG to isolate the CA3 from the CA1 and the DG. The second cut was along the hippocampal fissure to separate CA1 from DG. Total RNA was isolated from 3–6 slices or 3–10 microdissected regions (CA1, CA3 or DG) of slices using TriReagent (Molecular Research Center, Cincinnati, OH, USA). First strand cDNA was made in 20 ml using MuLV RT (Perkin Elmer Cetus, NJ, USA) and random hexamers, then amplified, five cycles of (94°C, 30 s; 48°C, 30 s; ramp to 72°C, 1 min 10 s; 72°C, 30 s), 35 cycles of (94°C, 30 s; 53°C, 30 s; 72°C, 30 s) using the Perkin Elmer PCR kit, AmpliTaq DNA polymerase and 100 nM oligonucleotide primers [2] which amplify NR2A, NR2B and NR2C subunits, producing products each 547 bp. Three enzymes, BpmI, BfaI, and ScaI, which selectively cut NR2A, NR2B and NR2C, respectively, [2], were used to digest one-third of the PCR product and produced restriction fragments of 321 and 226 bp, 392 and 155 bp, and 361 and 186 bp for NR2A, NR2B and NR2C, respectively (Fig. 1A). The restriction digest products were electrophoresed in a 1.5% agarose gel and visualized with ethidium bromide (Fig. 1A). The relative proportion of NR2 subunit mRNAs was determined by first measuring the intensities of the 321 bp fragment of the BpmI digest, the 392 bp fragment of the BfaI digest, and the 361 bp fragment of the ScaI digest, then measuring the intensities of the corresponding uncut fragments of each digest. The ratio of the intensity of the cut fragment to intensity of the uncut fragment for each subunit was calculated and expressed as a percent of the total amount of all three NR2 subunit mRNAs. The normal, relative proportions of NR2A and NR2B subunit mRNAs in whole, non-deprived hippocampal slices are 55.9 ± 3.6% (n = 8) and 39.0 ± 4.4% (n = 8), respectively, while NR2C subunit mRNA represents only 5.1 ± 2.2% (n = 8) of the total NR2 mRNA. The relative proportions of NR2A, NR2B and NR2C subunit mRNAs in microdissected CA1, CA3 and DG regions of the hippocampus 61.4 ± 5.3% (n = 14), 50.6 ± 5.3% (n = 7) and 38.2 ± 2.3% (n = 7), respectively for NR2A subunit mRNA, 35.6 ± 4.9% (n = 14), 45.0 ± 4.7% (n = 7) and 54.6 ± 3.9% (n = 7), respectively for NR2B subunit mRNA, and
Fig. 1. The expression of NR2A, NR2B and NR2C subunits in rat hippocampal slices before and at various times after the 4 min oxygen-glucose deprivation. (A) Example of the digestion products of the RT-PCR amplification of NR2 subunit mRNAs of a non-deprived (top) and deprived (bottom), whole hippocampal slice run in a 1.5% agarose gel. Digestion of the 547 bp product (uncut) with restriction enzymes, BpmI, BfaI, and ScaI, which selectively cut NR2A, NR2B and NR2C, respectively, results in fragments (cut) of 226 and 321 bp (lane A), 392 and 155 bp (lane B), and 361 and 186 bp (lane C). Often, as shown, only the larger of the cut fragments was clearly visible. (B) Change in the relative proportion of NR2 subunit expression in the slice following the 4 min oxygen-glucose deprivation. Means and SEM are shown for NR2A (O), NR2B (•) and NR2C (B) subunits. The number of samples assayed to obtain measures were 8, 6, 6 and 8 for times 0–90 min, respectively. The asterisk represents a significant (P , 0.05) difference between the preceeding time points and 90 min after re-oxygenation and return to 10 mM glucose for NR2C subunit expression as tested with an ANOVA.
D.L. Small et al. / Neuroscience Letters 232 (1997) 87–90
3.0 ± 1.0% (n = 14), 4.4 ± 1.8% (n = 7), and 2.9 ± 1.3% (n = 7), respectively for NR2C subunit mRNA. Following a 4 min deprivation of oxygen and glucose, the relative proportions of the NR2 subunit mRNAs in the whole hippocampal slice changes (Fig. 1B). Ninety minutes after the deprivation there is a significant (P , 0.05) increase in NR2C subunit expression from 5.1 ± 2.2% (n = 8) (non-deprived) to 20.2 ± 2.4% (n = 8) (deprived). This change in the relative NR2C subunit expression is consistent with Perez-Velazquez and Zhang’s [12] findings that NR2C subunit expression increased rapidly after the deprivation insult in the whole slice. Although the 4-fold increase in NR2C subunit mRNA was accompanied temporally by a decrease in NR2B subunit mRNA, the decrease was not significant (P . 0.05) (Fig. 1B). Likewise there was no significant (P . 0.05) change in NR2A subunit mRNA within the 90 min after re-oxygenation and return to 10 mM glucose. (Fig. 1B). To localize the changes in NR2C subunit mRNA in the hippocampal slice 90 min after oxygen-glucose deprivation, the vulnerable CA1, and resistant CA3 and DG regions of the hippocampus were microdissected, and the relative abundance of NR2 subunits were quantified. As in the whole slice, when non-deprived control tissues were compared to tissue 90 min after a deprivation insult, there was no significant (P . 0.05) change in the relative amount of NR2A or NR2B subunit expression within any of the microdissected regions of the hippocampus (Fig. 2A,B). On the other hand, in the vulnerable CA1 region, NR2C subunit expression increased significantly (P , 0.05), from 3.0 ± 1.0% (n = 14) to 10.8 ± 3.2% (n = 9) (Fig. 2C). Likewise, in the resistant CA3 and DG regions, there was a significant increase (P , 0.05) (Fig. 2C), from 4.4 ± 1.8% (n = 7) and 2.9 ± 1.3% (n = 7) to 15.1 ± 3.8% (n = 6) and 10.4 ± 3.2% (n = 6), respectively. The reported increase in NR2C subunit expression in the vulnerable CA1 region of hippocampal slices deprived of oxygen and glucose [18] is relevant to the development of NMDA antagonists for the treatment of stroke since the NR2C subunit is normally absent in the adult hippocampus [9] and hence, NR2C subunits could represent a novel therapeutic target for post-ischemic neurodegeneration. However, the role of NR2C subunits in CA1 neuronal degeneration is unclear. Although the weaker Mg2+ channel block of NR2C [9] might confer vulnerability, given that NR2C subunits are associated with a smaller single channel conductance and shorter mean open time [4]. Alternatively, the expression of NR2C subunits might instead confer resistance since expression of NR2A and NR2B subunits in HEK 293 cells enhances vulnerability [1,14], whereas cells with NR2C subunits were not compromised [1]. Also, during development when the hippocampus is relatively resistant to excitotoxicity [3], NR2B subunit expression is low and NR2C subunit expression is high [9,13], while in the cerebellum, a transient, vulnerability is coincident with NR2B subunit expression [5,6]. Our results,
89
Fig. 2. Relative NR2A (A), NR2B (B) and NR2C (C) subunit expression (mean ± SEM) in the CA1, CA3 and DG regions of the hippocampus before (control) and 90 min after (deprived) the 4 min deprivation insult. Significant differences between control and deprived slices within each region, as measured with an ANOVA, are represented by asterisks (P , 0.05). The number of samples assayed to obtain measures were 14, 7, and 7 for control and 9, 6 and 6 for deprived regions, respectively.
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obtained using the same ischemia model as Perez-Velazquez and Zhang [12] demonstrate that post-ischemic NR2C subunit induction in vitro is not restricted to the vulnerable CA1 region of the hippocampus, but expression is also significantly increased in the resistant CA3 and DG regions. This regional lack of specificity suggests that increased NR2C subunit expression does not contribute to the lethality of cerebral ischemia as postulated by PerezVelazquez and Zhang, [12]. Thus, the relative increase in NR2C subunit expression may be coincident with ischemia. Although the altered expression occurs uniformly throughout the hippocampus, the selective vulnerability exhibited by the CA1 region could be due to it’s inability to cope with this change, unlike the resistant CA3 and DG regions. Alternatively, given that NR2C subunit expression in the normal adult hippocampus is restricted to glial cells [17], the increase we observed could be occurring in glial cells. To understand these observations and their consequences to neuronal survival we have begun to examine the relative NR2 subunit mRNA in specific hippocampal cell types and to characterize their pathophysiology. This work was supported in part by the National Research Council of Canada and a grant (#ST2717) to P.M. and A.M.B. from the Heart and Stroke Foundation of Ontario (HSFO). We would like to thank Dr. Robert Monette for his expert assistance in the preparation of this manuscript. [1] Anegawa, N.J., Lynch, D.R., Verdoorn, T.A. and Pritchett, D.B., Transfection of N-methyl-d-aspartate receptors in a nonneuronal cell line leads to cell death, J. Neurochem., 64 (1995) 2004–2012. [2] Audinat, E., Lambolez, B., Rossier, J. and Cre´pel, F., Activitydependent regulation of N-methyl-d-aspartate receptor subunit expression in rat cerebellar granule cells, Eur. J. Neurosci., 6 (1994) 1792–1800. [3] Dunwiddie, T.V., Age-related differences in the in vitro rat hippocampus. Development of inhibition and the effects of hypoxia, Dev. Neurosci., 4 (1981) 165–175. [4] Ebralidze, A.K., Rossi, D.J., Tonegawa, S. and Slater, N.T., Modification of NMDA receptor channels and synaptic transmission by targeted disruption of the NR2C gene, J. Neurosci., 16 (1996) 5014–5025.
[5] Farrant, M., Feldmeyer, D., Takahashi, T. and Cull-Candy, S.G., NMDA-receptor channel diversity in the developing cerebellum, Nature, 368 (1994) 335–339. [6] Garthwaite, G. and Garthwaite, J., In vitro neurotoxicity of excitatory acid analogues during cerebellar development, Neuroscience, 17 (1986) 755–767. [7] Gass, P., Muelhardt, C., Sommer, C., Becker, C.-M. and Kiessling, M., NMDA and glycine receptor mRNA expression following transient global ischemia in the gerbil brain, J. Cerebral Blood Flow Metab., 13 (1993) 337–341. [8] Heurteaux, C., Lauritzen, I., Widmann, C. and Lazdunski, M., 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 (1994) 67–74. [9] Monyer, H., Brunashev, N., Laurie, D.J., Sakmann, B. and Seeburg, P.H., Developmental and regional expression in the rat brain and functional properties of four NMDA receptors, Neuron, 12 (1994) 529–540. [10] Muir, K.W. and Lees, K.R., Clinical experience with excitatory amino acid antagonist drugs, Stroke, 26 (1995) 503–513. [11] Pellegrini-Giampietro, D.E., Pulsinelli, W.A. and Zukin, R.S., NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists, J. Neurochem., 62 (1994) 1067–1073. [12] Perez-Velazquez, J.L. and Zhang, L., In vitro hypoxia induces expression of the NR2C subunit of the NMDA receptor in rat cortex and hippocampus, J. Neurochem., 63 (1994) 1171–1173. [13] Pollard, H., Khrestchatisky, M., Moreau, J. and Ben-Ari, Y., Transient expression of the NR2C subunit of the NMDA receptor in developing brain, NeuroReport, 4 (1993) 411–414. [14] Raymond, L.A., Moshaver, A., Tingley, W.G. and Huganir, R.L., Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line, Mol. Cell. Neurosci., 7 (1996) 102–115. [15] Sucher, N.J., Awobuluyi, M., Choi, Y.-B. and Lipton, S.A., NMDA receptors: from genes to channels, Trends Pharmacol. Sci., 17 (1996) 348–355. [16] Sugimoto, A., Takeda, A., Kogure, K. and Onodera, H., NMDA receptor (NMDAR1) expression in the rat hippocampus after forebrain ischemia, Neurosci. Lett., 170 (1994) 39–42. [17] Watanabe, M., Inoue, Y., Sakimura, K. and Mishina, M., Distinct distributions of five N-methyl-d-aspartate receptor channel subunit mRNAs in the forebrain, J. Comp. Neurol., 338 (1993) 377–390. [18] Zhang, L., Miu, P. and Eubanks, J.H., NMDA channel activities in rat CA1 hippocampal neurones following a brief hypoxic-hypoglycaemic challenge in brain slices, Soc. Neurosci. Abstr., 21 (1995) 392.1.
Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices Daniel L. Small a ,*, Michael O. Poulter a, Alastair M. Buchan b, Paul Morley a a
Institute for Biological Sciences, National Research Council of Canada, Building M-54, Montreal Road, Ottawa, Ontario, K1A 0R6 Canada b Foothills Hospital, 1403-29 Street N.W., Calgary, Alberta, T2N 2T9 Canada Received 4 June 1997; received in revised form 28 July 1997; accepted 29 July 1997
Abstract Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-d-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions. 1997 Elsevier Science Ireland Ltd. Keywords: In vitro ischemia; Excitotoxicity; Reverse transcription-polymerase chain reaction; N-Methyl-d-aspartate receptor; Hippocampus
Given that N-methyl-d-aspartate (NMDA) receptors are important targets of therapeutic intervention [10], and their pharmacological diversity is largely determined by the NR2 subunits of the heteromeric receptor [15], it is relevant to characterize the post-ischemic expression of NR2 subunits to ensure optimal specificity and efficacy of potential neuroprotective compounds. Although several studies have demonstrated changes in NMDA receptor subunit expression following cerebral ischemia [7,8,11,16], there is controversy associated with these changes. Some evidence suggests that there is no change in NR1 expression [7,11, 16], while other evidence suggests that there is a significant increase [8] and in situ hybridization studies suggest there is an increase in NR2A, and NR2B, but not NR2C [8]. In an in vitro hippocampal slice model of cerebral ischemia, PerezVelazquez and Zhang [12] observed a rapid (,45 min) increase in NR2C subunit mRNA expression which, based on preliminary results, also occurred in vivo [12]. Function-
* Corresponding author. Tel.:+1 613 9900902; fax: +1 613 9414475; e-mail: [email protected]
ally, using the in vitro model, Zhang et al. [18] reported a reduced Mg2+ sensitivity of NMDA-mediated currents, a biophysical feature of NR2C-containing receptors [9], in the vulnerable CA1 pyramidal neurons following 18 h of re-oxygenation and return to 4 mM glucose in the hippocampal slice. It is not known whether the increased NR2C expression is restricted to the vulnerable CA1 region of the hippocampus or whether NR2C induction confers resistance or vulnerability. Using reverse transcription-polymerase chain reaction (RT-PCR) amplification we have quantified the relative expression of NR2A-C subunits in rat hippocampal slices exposed to the same oxygen-glucose deprivation insult used by Perez-Velazquez and Zhang [12]. By quantifying the relative NR2 subunit expression in microdissected CA1, CA3 and dentate gyrus (DG) regions of hippocampal slices, we have localized the changes in NR2C expression to resistant and vulnerable regions of the hippocampus. Male Wistar rats (175–200 g) anesthetized with 3% isoflurane were decapitated and their brains were rapidly (,1 min) removed and submerged in cold (4°C), artificial cerebrospinal fluid (ACSF) consisting of (in mM): 127 NaCl, 2
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0592-2
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KCl, 10 glucose, 1.2 KH2PO4, 26 NaH2CO3, 2 MgSO4, 2 CaCl2 (pH 7.4) bubbled with 95% O2/5% CO2. Transverse hippocampal slices (400 mm) were placed onto floating nylon mesh platforms in covered incubation chambers (750 ml) such that the slices were submerged in 2–3 mm of 95% O2/5% CO2 bubbled ACSF at 34°C. After 90 min, slices that were to be deprived of oxygen and glucose were transferred to smaller chambers containing 30–40 ml of 95% N2/5% CO2 bubbled ACSF with 0 mM glucose for 4 min at 34°C. The temperature, glucose concentration, and duration of insult, were chosen to replicate the insult conditions of Perez-Velazquez and Zhang, [12]. Slices were returned to the 750 ml chamber following the 4 min insult. The CA1, CA3 and DG regions were microdissected in ACSF at 4°C using a 60 × dissecting microscope, fine forceps and a #15 scalpel blade. The first cut was perpendicular to the open, lateral end of the DG to isolate the CA3 from the CA1 and the DG. The second cut was along the hippocampal fissure to separate CA1 from DG. Total RNA was isolated from 3–6 slices or 3–10 microdissected regions (CA1, CA3 or DG) of slices using TriReagent (Molecular Research Center, Cincinnati, OH, USA). First strand cDNA was made in 20 ml using MuLV RT (Perkin Elmer Cetus, NJ, USA) and random hexamers, then amplified, five cycles of (94°C, 30 s; 48°C, 30 s; ramp to 72°C, 1 min 10 s; 72°C, 30 s), 35 cycles of (94°C, 30 s; 53°C, 30 s; 72°C, 30 s) using the Perkin Elmer PCR kit, AmpliTaq DNA polymerase and 100 nM oligonucleotide primers [2] which amplify NR2A, NR2B and NR2C subunits, producing products each 547 bp. Three enzymes, BpmI, BfaI, and ScaI, which selectively cut NR2A, NR2B and NR2C, respectively, [2], were used to digest one-third of the PCR product and produced restriction fragments of 321 and 226 bp, 392 and 155 bp, and 361 and 186 bp for NR2A, NR2B and NR2C, respectively (Fig. 1A). The restriction digest products were electrophoresed in a 1.5% agarose gel and visualized with ethidium bromide (Fig. 1A). The relative proportion of NR2 subunit mRNAs was determined by first measuring the intensities of the 321 bp fragment of the BpmI digest, the 392 bp fragment of the BfaI digest, and the 361 bp fragment of the ScaI digest, then measuring the intensities of the corresponding uncut fragments of each digest. The ratio of the intensity of the cut fragment to intensity of the uncut fragment for each subunit was calculated and expressed as a percent of the total amount of all three NR2 subunit mRNAs. The normal, relative proportions of NR2A and NR2B subunit mRNAs in whole, non-deprived hippocampal slices are 55.9 ± 3.6% (n = 8) and 39.0 ± 4.4% (n = 8), respectively, while NR2C subunit mRNA represents only 5.1 ± 2.2% (n = 8) of the total NR2 mRNA. The relative proportions of NR2A, NR2B and NR2C subunit mRNAs in microdissected CA1, CA3 and DG regions of the hippocampus 61.4 ± 5.3% (n = 14), 50.6 ± 5.3% (n = 7) and 38.2 ± 2.3% (n = 7), respectively for NR2A subunit mRNA, 35.6 ± 4.9% (n = 14), 45.0 ± 4.7% (n = 7) and 54.6 ± 3.9% (n = 7), respectively for NR2B subunit mRNA, and
Fig. 1. The expression of NR2A, NR2B and NR2C subunits in rat hippocampal slices before and at various times after the 4 min oxygen-glucose deprivation. (A) Example of the digestion products of the RT-PCR amplification of NR2 subunit mRNAs of a non-deprived (top) and deprived (bottom), whole hippocampal slice run in a 1.5% agarose gel. Digestion of the 547 bp product (uncut) with restriction enzymes, BpmI, BfaI, and ScaI, which selectively cut NR2A, NR2B and NR2C, respectively, results in fragments (cut) of 226 and 321 bp (lane A), 392 and 155 bp (lane B), and 361 and 186 bp (lane C). Often, as shown, only the larger of the cut fragments was clearly visible. (B) Change in the relative proportion of NR2 subunit expression in the slice following the 4 min oxygen-glucose deprivation. Means and SEM are shown for NR2A (O), NR2B (•) and NR2C (B) subunits. The number of samples assayed to obtain measures were 8, 6, 6 and 8 for times 0–90 min, respectively. The asterisk represents a significant (P , 0.05) difference between the preceeding time points and 90 min after re-oxygenation and return to 10 mM glucose for NR2C subunit expression as tested with an ANOVA.
D.L. Small et al. / Neuroscience Letters 232 (1997) 87–90
3.0 ± 1.0% (n = 14), 4.4 ± 1.8% (n = 7), and 2.9 ± 1.3% (n = 7), respectively for NR2C subunit mRNA. Following a 4 min deprivation of oxygen and glucose, the relative proportions of the NR2 subunit mRNAs in the whole hippocampal slice changes (Fig. 1B). Ninety minutes after the deprivation there is a significant (P , 0.05) increase in NR2C subunit expression from 5.1 ± 2.2% (n = 8) (non-deprived) to 20.2 ± 2.4% (n = 8) (deprived). This change in the relative NR2C subunit expression is consistent with Perez-Velazquez and Zhang’s [12] findings that NR2C subunit expression increased rapidly after the deprivation insult in the whole slice. Although the 4-fold increase in NR2C subunit mRNA was accompanied temporally by a decrease in NR2B subunit mRNA, the decrease was not significant (P . 0.05) (Fig. 1B). Likewise there was no significant (P . 0.05) change in NR2A subunit mRNA within the 90 min after re-oxygenation and return to 10 mM glucose. (Fig. 1B). To localize the changes in NR2C subunit mRNA in the hippocampal slice 90 min after oxygen-glucose deprivation, the vulnerable CA1, and resistant CA3 and DG regions of the hippocampus were microdissected, and the relative abundance of NR2 subunits were quantified. As in the whole slice, when non-deprived control tissues were compared to tissue 90 min after a deprivation insult, there was no significant (P . 0.05) change in the relative amount of NR2A or NR2B subunit expression within any of the microdissected regions of the hippocampus (Fig. 2A,B). On the other hand, in the vulnerable CA1 region, NR2C subunit expression increased significantly (P , 0.05), from 3.0 ± 1.0% (n = 14) to 10.8 ± 3.2% (n = 9) (Fig. 2C). Likewise, in the resistant CA3 and DG regions, there was a significant increase (P , 0.05) (Fig. 2C), from 4.4 ± 1.8% (n = 7) and 2.9 ± 1.3% (n = 7) to 15.1 ± 3.8% (n = 6) and 10.4 ± 3.2% (n = 6), respectively. The reported increase in NR2C subunit expression in the vulnerable CA1 region of hippocampal slices deprived of oxygen and glucose [18] is relevant to the development of NMDA antagonists for the treatment of stroke since the NR2C subunit is normally absent in the adult hippocampus [9] and hence, NR2C subunits could represent a novel therapeutic target for post-ischemic neurodegeneration. However, the role of NR2C subunits in CA1 neuronal degeneration is unclear. Although the weaker Mg2+ channel block of NR2C [9] might confer vulnerability, given that NR2C subunits are associated with a smaller single channel conductance and shorter mean open time [4]. Alternatively, the expression of NR2C subunits might instead confer resistance since expression of NR2A and NR2B subunits in HEK 293 cells enhances vulnerability [1,14], whereas cells with NR2C subunits were not compromised [1]. Also, during development when the hippocampus is relatively resistant to excitotoxicity [3], NR2B subunit expression is low and NR2C subunit expression is high [9,13], while in the cerebellum, a transient, vulnerability is coincident with NR2B subunit expression [5,6]. Our results,
89
Fig. 2. Relative NR2A (A), NR2B (B) and NR2C (C) subunit expression (mean ± SEM) in the CA1, CA3 and DG regions of the hippocampus before (control) and 90 min after (deprived) the 4 min deprivation insult. Significant differences between control and deprived slices within each region, as measured with an ANOVA, are represented by asterisks (P , 0.05). The number of samples assayed to obtain measures were 14, 7, and 7 for control and 9, 6 and 6 for deprived regions, respectively.
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D.L. Small et al. / Neuroscience Letters 232 (1997) 87–90
obtained using the same ischemia model as Perez-Velazquez and Zhang [12] demonstrate that post-ischemic NR2C subunit induction in vitro is not restricted to the vulnerable CA1 region of the hippocampus, but expression is also significantly increased in the resistant CA3 and DG regions. This regional lack of specificity suggests that increased NR2C subunit expression does not contribute to the lethality of cerebral ischemia as postulated by PerezVelazquez and Zhang, [12]. Thus, the relative increase in NR2C subunit expression may be coincident with ischemia. Although the altered expression occurs uniformly throughout the hippocampus, the selective vulnerability exhibited by the CA1 region could be due to it’s inability to cope with this change, unlike the resistant CA3 and DG regions. Alternatively, given that NR2C subunit expression in the normal adult hippocampus is restricted to glial cells [17], the increase we observed could be occurring in glial cells. To understand these observations and their consequences to neuronal survival we have begun to examine the relative NR2 subunit mRNA in specific hippocampal cell types and to characterize their pathophysiology. This work was supported in part by the National Research Council of Canada and a grant (#ST2717) to P.M. and A.M.B. from the Heart and Stroke Foundation of Ontario (HSFO). We would like to thank Dr. Robert Monette for his expert assistance in the preparation of this manuscript. [1] Anegawa, N.J., Lynch, D.R., Verdoorn, T.A. and Pritchett, D.B., Transfection of N-methyl-d-aspartate receptors in a nonneuronal cell line leads to cell death, J. Neurochem., 64 (1995) 2004–2012. [2] Audinat, E., Lambolez, B., Rossier, J. and Cre´pel, F., Activitydependent regulation of N-methyl-d-aspartate receptor subunit expression in rat cerebellar granule cells, Eur. J. Neurosci., 6 (1994) 1792–1800. [3] Dunwiddie, T.V., Age-related differences in the in vitro rat hippocampus. Development of inhibition and the effects of hypoxia, Dev. Neurosci., 4 (1981) 165–175. [4] Ebralidze, A.K., Rossi, D.J., Tonegawa, S. and Slater, N.T., Modification of NMDA receptor channels and synaptic transmission by targeted disruption of the NR2C gene, J. Neurosci., 16 (1996) 5014–5025.
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