- Email: [email protected]
Cortical NMDAR-1 gene expression is rapidly upregulated after seizure
Molecular Brain Research 44 Ž1997. 157–162
Short communication
Cortical NMDAR-1 gene expression is rapidly upregulated after seizure Penny J. Jensen
a,c
, Noemi Millan c , Kenneth J. Mack
a,b,c,)
a
Department of Physiology, UniÕersity of Wisconsin, Madison, WI, USA Department of Neurology, UniÕersity of Wisconsin, Madison, WI, USA Waisman Center on Mental Retardation, UniÕersity of Wisconsin, Madison, WI, USA b
c
Accepted 1 October 1996
Abstract The promoter region of the NMDAR-1 receptor has a cis-regulatory element that is capable of binding to the NGFI-A family of transcription factors. Based on this observation, we hypothesized that situations that cause a change in NGFI-A levels would result in a change in NMDAR-1 expression. In these studies, we have demonstrated that a seizure results in a rapid significant increase in NMDAR-1 mRNA and protein expression, at a time when NGFI-A protein levels are expected to be elevated. Our results indicate that control of NMDAR-1 expression is stimulus, time and tissue dependent. Keywords: Glutamate receptor; NMDAR-1; NGFI-A; Early growth response ŽEGR.; Transcription; Pentylenetetrazol
The glutamate receptor NMDAR-1 Ž N-methyl-D-aspartate R-1. is thought to participate in the establishment and maintenance of synaptic plasticity underlying learning and memory. The 5X-flanking region of the rat NMDAR-1 gene has been cloned w1x. Analysis of the promoter region of the NMDAR-1 gene has shown that the proximal y356 bp to the transcription initiation site contains the essential elements to promote neuron-specific expression w1x. Footprint demonstrations depict a 112-bp region that is protected upon DNase I digestion w2x. Within this region resides a GSG box. The GSG box is a 9-bp sequence ŽGCGGGGGCG. w7x that is recognized by the EGR Žearly growth response. family of immediate-early genes, of which NGFI-A is a member. These observations suggest that NGFI-A, or another member of the EGR family, plays a role in the transcriptional regulation of the NMDAR-1 gene. We hypothesized that, if NGFI-A Žor a related EGR family member. affects NMDAR-1 expression, then we should observe changes in the levels of NMDAR-1 mRNA at a time when the protein levels of these transcription factors are expected to be elevated. Previous data describes the time point for NGFI-A and related protein induction as
) Corresponding author. Waisman Center, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705, USA. Fax: q1 Ž608. 265-4103.
1–2 h post-stimulation w14x. These studies used a model of experience-dependent plasticity Žwhisker stimulation; w14x. as well as pentylenetetrazol ŽPTZ.-induced seizures to study this possible relationship. Results from this study show that there are specific regional-, temporal- and stimulation-dependent alterations in the expression of NMDAR-1 mRNA and protein. Adult Ž200–300 g. male albino Sprague-Dawley rats were used in these studies. The induction of PTZ-induced seizures and whisker stimulation was as previously described w14x. In situ hybridization analysis was performed on 30-m m free-floating sections according to Esclapez et al. w8x. cRNA probes were produced and labeled with either digoxigenin or P 33 and represented bp 1940–2646 of the NMDAR-1 receptor mRNA w18x. For RT-PCR experiments, 2 m g total RNA, isolated either by the Chomczynski and Sacchi method w6x or an RNA isolation kit from Pharmacia, was used in a RT-PCR reaction. Primers were designed to detect all known splice variants of NMDAR-1, encompassing bp 1940–2646 and yielding a product of 714 bp. PCR cycles were as follows: 958C 5 min, 26 cycles of 1 min 958C, 1.5 min 618C, 2.5 min 728C and ended by 6 min 728C. These cycles were optimized and verified to be within the linear range of amplification. PCR products were visualized on a 12% polyacrylamide gel through the incorporation of P 33 alpha into the PCR reaction. These gels were examined by drying and exposing to x-ray film and then quantifying
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 6 . 0 0 2 6 2 - 8
158
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
through densitometry using NIH IMAGE analysis software. Similar results were obtained by cutting out corresponding gel slices and counting them in a scintillation counter. Rabbit anti-NMDAR-1 antibody was purchased from Chemicon International ŽAb1516; Temecula, CA, USA. and is selective for splice variants NR1-1a, NR1-1b, NR12a and NR1-2b. Crude membrane preparations were prepared following the methods of Brose et al. w4x. 30 m g of crude membranes were loading onto a 8.5% SDS-PAGE gel. The proteins were then electroblotted to nylon membranes ŽN-2519; Sigma.. The resulting Western blot was blocked with 3% BSArTBS for 1 h and exposed to the primary NMDAR-1 antibody at 0.5 m grml for 3 h, then a 1 : 12500 secondary antibody solution for 2 h. Detection was achieved with Lumiphos ŽBoehringer Mannheim. and exposed to Kodak X-OMAT film. Analysis of bands was through densitometry using the NIH image software program. Our laboratory has been interested in the transcriptional regulation of neuronal gene expression in the somatosensory cortex, using models of experience-dependent plasticity and seizures. Our initial studies asked the question of
whether these stimuli affected somatosensory cortex expression of NMDAR-1 mRNA. In situ hybridization using either a digoxigenin or P 33 -labeled antisense NMDAR-1 probe was performed on rat brain slices following either a seizure or physiological stimulation paradigm. Animals were exposed to 50 mgrkg of i.p. PTZ, which resulted in a brief motor seizure. In other groups of animals, 1.1 grkg of urethane was used to produce a light anesthesia so that an animal could have its whiskers stimulated by an artist’s paintbrush for 15 min. This model attempts to duplicate a sensory whisker experience that an animal may experience naturally w14x. A total of 3 animalsrtime periodrstimulation paradigm were analyzed. As would be expected, the mRNA transcript for NMDAR-1 is quite ubiquitous but most remarkable is an apparent increase in NMDAR-1 message in the motor cortex after a PTZ-induced seizure ŽFig. 1.. Contrary to our initial expectations, no increase was detectable in the somatosensory cortex area or hippocampus and this induction was specific for the motor cortex ŽFig. 2.. When labeled sense probes were used as controls, there was no display of staining. Intense increases of NMDAR-1 mRNA-staining were seen 1 and 2 h postPTZ-induced seizure. High magnification of the motor
Fig. 1. NMDAR-1 mRNA abundance in the cortex after a seizure experience under high magnification. Analysis of NMDAR-1 mRNA expression by in situ hybridization using a digoxigenin antisense-labeled probe. These photomicrographs illustrate that NMDAR-1 mRNA staining is elevated at the 1 h time-period post-PTZ-induced seizure compared to the control animal. These results also demonstrate that the increase in NMDAR-1 abundance occurs in all layers of the cortex. Labeled sense probe as a control shows no staining.
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
cortex after seizure induction illustrates that the increase in expression appears to be widespread throughout layers II-VI of the cortex ŽFig. 1.. To further quantify the expression and induction of NMDAR-1 mRNA, RT-PCR analysis was performed on brain tissue isolated from rats sacrificed at specific times post either PTZ injection or physiological stimulation. Representative densitometric data from NMDAR-1 RTPCR analysis on 3 different brain regions: motor cortex, somatosensory cortex and hippocampus after a PTZ-induced seizure are illustrated in Fig. 3. In the motor cortex, there is a significant Ž P - 0.005; Student’s t-test. difference in the abundance of NMDAR-1 mRNA 2 h post-PTZ seizure induction compared to control. Parallel results were obtained by scintillation counting the gel slices as by densitometric analysis of the autoradiographs of the gels. In the motor, somatosensory cortex and hippocampus regions, there is a slight, but non-significant, upregulation of NMDAR-1 mRNA 1 h after seizure activity Ždata not shown.. Following whisker brushing, there is not a significant alteration in NMDAR-1 levels from the contralateral Žstimulated cortex. to the ipsilateral Žcontrol. cortex. This illustrates that physiological stimulation does not significantly alter NMDAR-1 expression within 4 h post-stimulation.
159
In order to determine if changes in NMDAR-1 mRNA levels will result in changes in the amount of NMDAR-1 protein, Western blots were performed. These results demonstrate that PTZ evoked seizures elevate the levels of NMDAR-1 protein in the motor cortex 1 and 2 h after seizure activity ŽFig. 4.. Densitometric values taken from the autoradiographs demonstrate a significant Ž P - 0.005. increase between control and the 1- and 2-h time points. With a significant decrease, G 6 h times. The somatosensory cortex exhibits no change in protein levels Žnot shown.. This study illustrates that seizure activity alters the expression of NMDAR-1 in the motor cortex at both the mRNA and protein levels. These changes occur at times Ž1–2 h after stimulation. when increased levels of NGFI-A protein are expected to be seen w13x. These data support the hypothesis that NGFI-A Žor related factors. may play a role in the regulation of NMDAR-1 transcription although other tissue-specific factors seem to be involved as well. Previous data from this laboratory demonstrates that levels of expression of certain EGRs, such as NGFI-A w14x, NGFI-C w16x and Krox 20 w15x, are highly dissimilar following different stimulation paradigms. For example, NGFI-A or NGFI-C mRNA levels increase by 80% consequent to a physiological vibrissae stimulation while in-
Fig. 2. Staining of NMDAR-1 subunit mRNA post-1 h PTZ-induced seizure is specific to the motor cortex. These photomicrographs illustrate that the increase in NMDAR-1 receptor subunit is specifically orientated to the motor cortex.
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P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
creasing ) 400% after a seizure stimulus w14x. This induction of mRNA reaches maximum levels by 30–60 min after the stimulus. Protein levels of NGFI-A are also increased subsequent to a PTZ-induced seizure starting at 1 h post-stimulus and remaining elevated for at least 4 h w13x. These observations suggest that, if NGFI-A Žor a related member of the EGR family. affects NMDAR-1 expression, then a robust increase, such as is seen after a seizure, in these transcription factors would be needed to affect transcription of NMDAR-1. Why would the motor cortex be specifically affected? This phenomenon could be caused by the specific agent employed for seizure induction. PTZ produces a generalized seizure which does not specifically require afferent information to be carried to the somatosensory cortex.
Secondly, it is possible that specific subregions of the neocortex possess an array of mechanisms available to modify the effectiveness of its synapses. For example, the induction of LTP in the cortex appears to be different between motor and sensory cortex w5x. Finally, a second molecular example of motorrsensory differences occurs in the songbird forebrain in response to PTZ. After PTZ, there occurs a dramatically higher increase in NGFI-A ŽZENK. expression in motor areas than in sensory areas w17x. In rat, NMDAR-1 expression appears to be neuronspecific and levels are altered following certain pathological or physiological conditions. For instance, in partially kindled animals, no change in NMDAR-1 mRNA expression was displayed, while after full kindling, NMDAR-1
Fig. 3. Investigation of the abundance of NMDAR-1 mRNA expression through RT-PCR. 2 m g of total RNA extracted from different tissue types after disparate stimulation paradigms were used in RT-PCR experiments. P 33 -labeled nucleotides were added to the PCR reaction to quantify the results. Preliminary experiments demonstrated that the PCR reactions were still in the linear amplification phase. The graph depicts densitometric values of the autoradiographs of the PCR gels. Numbers were normalized to a 100 control value. Results for motor cortex ŽMC. show a trend to increase both 1 and 2 h with the post-2 h increase being significant at the P - 0.005 value Ž n s 7.. After 2 h, the levels of NMDAR-1 mRNA fall but are still slightly above control values. Conclusions from somatosensory cortex ŽSSC. Ž n s 5. and hippocampus ŽHC. at the 2 h Ž n s 5. time post-seizure demonstrate that there are only negligible increases in mRNA expression. The same conclusions were reached for experiments with scintillation counting the PCR gels for interpretation of PCR changes.
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
161
Acknowledgements This work was supported by National Institute of Health Grant NS 33913 Žto K.J.M... We thank Amy Pletsch for her work in the preliminary stages of this project.
References
Fig. 4. Changes in levels of NMDAR-1 protein after seizure. Western blots were performed on disparate tissue samples to observe any alterations in NMDAR-1 protein levels following a PTZ-induced seizure. Effects from seizure are a significant increase in protein levels both 1 and 2 h post-seizure and significant decreases at 6 and 24 h after stimulation. This gel depicts a time course post-seizure stimulation specifically in the motor cortex. Proteins were detected through the use of Luma-Phos ŽBoehringer-Mannheim. and further analyzed by densitometry using the NIH image software.
mRNA abundance was downregulated specifically in the dentate gyrus granule cells with a maximum decrease 4 h post-stimulation with levels returned to normal within 12 h w20x. A different study, while using the same kindling process, found no change in NMDAR-1 message 24 h after the last stimulation w11x. No change was documented 24 h following both electroconvulsive stimulation ŽECS. w19x or kainate-induced seizure w9x. Stress brings about a 35–45% decrease in NMDAR-1 expression 24 h after induction w3x. Another example is a decrease in NMDAR-1 expression seen in rat spinal cord 7.5 h and 3 days subsequent to unilateral hind paw inflammation w12x. Collectively, these data suggest that NMDAR-1 is regulated in a stimulus-, temporal- and tissue-specific manner. The mechanisms underlying these alterations remain to be elucidated. Preliminary gel-shift experiments from our laboratory demonstrate that NGFI-A, NGFI-C and EGR-3 have the capability of binding to the promoter region of NMDAR-1 w10x. Preliminary co-transfection experiments from our laboratory w10x demonstrate that the EGR family of transcription factors does indeed bring about changes in the expression of a luciferase expression plasmid under the control of the NMDAR-1 5X-flanking region in PC12 cells. The above in vitro results indicate that the EGR family of transcription factors have the potential to affect NMDAR-1 gene expression. The results of this paper shows that increased levels of NMDAR-1 expression occurs at a time when high levels of NGFI-A protein are expected physiologically and therefore may play a role in NMDAR-1 gene regulation.
w1x Bai and Kusiak, Cloning and analysis of the 5X flanking sequence of the rat N-methyl-D-aspartate receptor 1 ŽNMDAR1. gene, Biochim. Biophys. Acta, 1152 Ž1993. 197–200. w2x Bai and Kusiak, Functional analysis of the proximal 5X-flanking region of the N-methyl-D-aspartate receptor subunit gene, NMDAR1, J. Biol. Chem., 270 Ž1995. 7737–7744. w3x Bartunsz, V., Aubry, J.M., Pagliusi, S., Jezova, D., Baffi, J. and Kiss, J.Z., Stress-induced changes in messenger RNA levels of N-methyl-D-aspartate and AMPA receptor subunits in selected regions of the rat hippocampus and hypothalamus, Neuroscience, 66 Ž1995. 247–252. w4x Brose, N., Gasic, G.P., Vetter, D.E., Sullivan, J.M. and Heinemann, S.F., Protein chemical characterization and immunocytochemical localization of the NMDA receptor subunit NMDAR1, J. Biol. Chem., 268 Ž1993. 22663–22671. w5x Castro-Alamancos, M.A., Donoghue, J.P. and Connors, B.W., Different forms of synaptic plasticity in somatosensory and motor areas of the neocortex, J. Neurosci., 15 Ž1995. 5324–5333. w6x Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 Ž1987. 156–159. w7x Christy, B. and Nathans, D., DNA binding site of the growth-factorinducible protein Zif268, Proc. Natl. Acad. Sci. USA, 86 Ž1989. 8737–8741. w8x Esclapez, M., Tillajaratbem, N.J.K., Tobin, A.J. and Houser, C.R., Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods, J. Comp. Neurol., 331 Ž1993. 339–362. w9x Friedmann, L.K., Pellegrini-Giampietro, D.E., Sperber, E.F., Bennett, M.V.L., Moshe, S.L. and Zukin, R.S., Kainate-induced status epilepticus alters glutamate and GABA A receptor gene expression in adult hippocampus: an in situ hybridization study, J. Neurosci., 14 Ž1994. 2697–2707. w10x Jensen, P.J., Mack, K.J. and Millan, N., NMDAR-1 is a potential target gene for the EGR family of transcriptional factors, Society for Neuroscience Abstract, number 238.8 Ž1996. p. 596. w11x Kamphuis, W., Hendriksen, H., Diegenback, P.C. and Lopes Da Silva, F.H., N-Methyl-D-aspartate and kainate receptor gene expression in hippocampal pyramidal and granular neurons in the kindling model of epileptogenesis, Neuroscience, 67 Ž1995. 551–559. w12x Kus, L., Sanderson, J. and Beitz, A.J., N-Methyl-D-aspartate R1 messenger RNA and w 125 IxMK-801 binding decrease in rat spinal cord after unilateral hind paw inflammation, Neuroscience, 68 Ž1995. 159–165. w13x Mack, K.J., Day, M., Milbrandt, J. and Gottlieb, D.I., Localization of the NGFI-A protein in rat brain, Mol. Brain Res., 8 Ž1990. 177–180. w14x Mack, K.J. and Mack, P.A., Induction of transcription factors in somatosensory cortex after tactile stimulation, Mol. Brain Res., 12 Ž1992. 141–147. w15x Mack, K.J., Cortner, J., Mack, P. and Farnham, P.J., krox 20 messenger RNA and protein expression in the adult central nervous system, Mol. Brain Res., 14 Ž1992. 117–123. w16x Mack, K.J., Yi, S.D., Chang, S., Millan, N. and Mack, P., NGFI-C expression is affected by physiological stimulation and seizures in the somatosensory cortex, Mol. Brain Res., 29 Ž1995. 140–146.
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w17x Mellow, C.V. and Clayton, D.F., Song-induced ZENK gene expression in Auditory pathways of songbird brain and its relation to the song control system, J. Neurosci., 14 Ž1994. 6652–6666. w18x Moriyoshi, K., Masayuki, M., Ishii, T., Shigemoto, R., Mizuno, N. and Nakanishi, S., Molecular cloning and characterization of the rat NMDA receptor, Nature, 354 Ž1991. 31–37. w19x Naylor, P., Stewart, C.A., Wright, S.R., Pearson, R.C.A. and Reid,
I.C., Repeated ECS induces Glur1 but not NMDAR1A-G mRNA in the rat hippocampus, Mol. Brain Res., 35 Ž1996. 349–353. w20x Pratt, G.D., Kokaia, M., Bengzon, J., Kokaia, Z., Fritschy, J.M., Mohler, H. and Lindvall, O., Differential regulation of N-methyl-Daspartate receptor subunit messenger RNAs in kindling-induced epileptogenesis, Neuroscience, 57 Ž1993. 307–318.
Short communication
Cortical NMDAR-1 gene expression is rapidly upregulated after seizure Penny J. Jensen
a,c
, Noemi Millan c , Kenneth J. Mack
a,b,c,)
a
Department of Physiology, UniÕersity of Wisconsin, Madison, WI, USA Department of Neurology, UniÕersity of Wisconsin, Madison, WI, USA Waisman Center on Mental Retardation, UniÕersity of Wisconsin, Madison, WI, USA b
c
Accepted 1 October 1996
Abstract The promoter region of the NMDAR-1 receptor has a cis-regulatory element that is capable of binding to the NGFI-A family of transcription factors. Based on this observation, we hypothesized that situations that cause a change in NGFI-A levels would result in a change in NMDAR-1 expression. In these studies, we have demonstrated that a seizure results in a rapid significant increase in NMDAR-1 mRNA and protein expression, at a time when NGFI-A protein levels are expected to be elevated. Our results indicate that control of NMDAR-1 expression is stimulus, time and tissue dependent. Keywords: Glutamate receptor; NMDAR-1; NGFI-A; Early growth response ŽEGR.; Transcription; Pentylenetetrazol
The glutamate receptor NMDAR-1 Ž N-methyl-D-aspartate R-1. is thought to participate in the establishment and maintenance of synaptic plasticity underlying learning and memory. The 5X-flanking region of the rat NMDAR-1 gene has been cloned w1x. Analysis of the promoter region of the NMDAR-1 gene has shown that the proximal y356 bp to the transcription initiation site contains the essential elements to promote neuron-specific expression w1x. Footprint demonstrations depict a 112-bp region that is protected upon DNase I digestion w2x. Within this region resides a GSG box. The GSG box is a 9-bp sequence ŽGCGGGGGCG. w7x that is recognized by the EGR Žearly growth response. family of immediate-early genes, of which NGFI-A is a member. These observations suggest that NGFI-A, or another member of the EGR family, plays a role in the transcriptional regulation of the NMDAR-1 gene. We hypothesized that, if NGFI-A Žor a related EGR family member. affects NMDAR-1 expression, then we should observe changes in the levels of NMDAR-1 mRNA at a time when the protein levels of these transcription factors are expected to be elevated. Previous data describes the time point for NGFI-A and related protein induction as
) Corresponding author. Waisman Center, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705, USA. Fax: q1 Ž608. 265-4103.
1–2 h post-stimulation w14x. These studies used a model of experience-dependent plasticity Žwhisker stimulation; w14x. as well as pentylenetetrazol ŽPTZ.-induced seizures to study this possible relationship. Results from this study show that there are specific regional-, temporal- and stimulation-dependent alterations in the expression of NMDAR-1 mRNA and protein. Adult Ž200–300 g. male albino Sprague-Dawley rats were used in these studies. The induction of PTZ-induced seizures and whisker stimulation was as previously described w14x. In situ hybridization analysis was performed on 30-m m free-floating sections according to Esclapez et al. w8x. cRNA probes were produced and labeled with either digoxigenin or P 33 and represented bp 1940–2646 of the NMDAR-1 receptor mRNA w18x. For RT-PCR experiments, 2 m g total RNA, isolated either by the Chomczynski and Sacchi method w6x or an RNA isolation kit from Pharmacia, was used in a RT-PCR reaction. Primers were designed to detect all known splice variants of NMDAR-1, encompassing bp 1940–2646 and yielding a product of 714 bp. PCR cycles were as follows: 958C 5 min, 26 cycles of 1 min 958C, 1.5 min 618C, 2.5 min 728C and ended by 6 min 728C. These cycles were optimized and verified to be within the linear range of amplification. PCR products were visualized on a 12% polyacrylamide gel through the incorporation of P 33 alpha into the PCR reaction. These gels were examined by drying and exposing to x-ray film and then quantifying
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 6 . 0 0 2 6 2 - 8
158
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
through densitometry using NIH IMAGE analysis software. Similar results were obtained by cutting out corresponding gel slices and counting them in a scintillation counter. Rabbit anti-NMDAR-1 antibody was purchased from Chemicon International ŽAb1516; Temecula, CA, USA. and is selective for splice variants NR1-1a, NR1-1b, NR12a and NR1-2b. Crude membrane preparations were prepared following the methods of Brose et al. w4x. 30 m g of crude membranes were loading onto a 8.5% SDS-PAGE gel. The proteins were then electroblotted to nylon membranes ŽN-2519; Sigma.. The resulting Western blot was blocked with 3% BSArTBS for 1 h and exposed to the primary NMDAR-1 antibody at 0.5 m grml for 3 h, then a 1 : 12500 secondary antibody solution for 2 h. Detection was achieved with Lumiphos ŽBoehringer Mannheim. and exposed to Kodak X-OMAT film. Analysis of bands was through densitometry using the NIH image software program. Our laboratory has been interested in the transcriptional regulation of neuronal gene expression in the somatosensory cortex, using models of experience-dependent plasticity and seizures. Our initial studies asked the question of
whether these stimuli affected somatosensory cortex expression of NMDAR-1 mRNA. In situ hybridization using either a digoxigenin or P 33 -labeled antisense NMDAR-1 probe was performed on rat brain slices following either a seizure or physiological stimulation paradigm. Animals were exposed to 50 mgrkg of i.p. PTZ, which resulted in a brief motor seizure. In other groups of animals, 1.1 grkg of urethane was used to produce a light anesthesia so that an animal could have its whiskers stimulated by an artist’s paintbrush for 15 min. This model attempts to duplicate a sensory whisker experience that an animal may experience naturally w14x. A total of 3 animalsrtime periodrstimulation paradigm were analyzed. As would be expected, the mRNA transcript for NMDAR-1 is quite ubiquitous but most remarkable is an apparent increase in NMDAR-1 message in the motor cortex after a PTZ-induced seizure ŽFig. 1.. Contrary to our initial expectations, no increase was detectable in the somatosensory cortex area or hippocampus and this induction was specific for the motor cortex ŽFig. 2.. When labeled sense probes were used as controls, there was no display of staining. Intense increases of NMDAR-1 mRNA-staining were seen 1 and 2 h postPTZ-induced seizure. High magnification of the motor
Fig. 1. NMDAR-1 mRNA abundance in the cortex after a seizure experience under high magnification. Analysis of NMDAR-1 mRNA expression by in situ hybridization using a digoxigenin antisense-labeled probe. These photomicrographs illustrate that NMDAR-1 mRNA staining is elevated at the 1 h time-period post-PTZ-induced seizure compared to the control animal. These results also demonstrate that the increase in NMDAR-1 abundance occurs in all layers of the cortex. Labeled sense probe as a control shows no staining.
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
cortex after seizure induction illustrates that the increase in expression appears to be widespread throughout layers II-VI of the cortex ŽFig. 1.. To further quantify the expression and induction of NMDAR-1 mRNA, RT-PCR analysis was performed on brain tissue isolated from rats sacrificed at specific times post either PTZ injection or physiological stimulation. Representative densitometric data from NMDAR-1 RTPCR analysis on 3 different brain regions: motor cortex, somatosensory cortex and hippocampus after a PTZ-induced seizure are illustrated in Fig. 3. In the motor cortex, there is a significant Ž P - 0.005; Student’s t-test. difference in the abundance of NMDAR-1 mRNA 2 h post-PTZ seizure induction compared to control. Parallel results were obtained by scintillation counting the gel slices as by densitometric analysis of the autoradiographs of the gels. In the motor, somatosensory cortex and hippocampus regions, there is a slight, but non-significant, upregulation of NMDAR-1 mRNA 1 h after seizure activity Ždata not shown.. Following whisker brushing, there is not a significant alteration in NMDAR-1 levels from the contralateral Žstimulated cortex. to the ipsilateral Žcontrol. cortex. This illustrates that physiological stimulation does not significantly alter NMDAR-1 expression within 4 h post-stimulation.
159
In order to determine if changes in NMDAR-1 mRNA levels will result in changes in the amount of NMDAR-1 protein, Western blots were performed. These results demonstrate that PTZ evoked seizures elevate the levels of NMDAR-1 protein in the motor cortex 1 and 2 h after seizure activity ŽFig. 4.. Densitometric values taken from the autoradiographs demonstrate a significant Ž P - 0.005. increase between control and the 1- and 2-h time points. With a significant decrease, G 6 h times. The somatosensory cortex exhibits no change in protein levels Žnot shown.. This study illustrates that seizure activity alters the expression of NMDAR-1 in the motor cortex at both the mRNA and protein levels. These changes occur at times Ž1–2 h after stimulation. when increased levels of NGFI-A protein are expected to be seen w13x. These data support the hypothesis that NGFI-A Žor related factors. may play a role in the regulation of NMDAR-1 transcription although other tissue-specific factors seem to be involved as well. Previous data from this laboratory demonstrates that levels of expression of certain EGRs, such as NGFI-A w14x, NGFI-C w16x and Krox 20 w15x, are highly dissimilar following different stimulation paradigms. For example, NGFI-A or NGFI-C mRNA levels increase by 80% consequent to a physiological vibrissae stimulation while in-
Fig. 2. Staining of NMDAR-1 subunit mRNA post-1 h PTZ-induced seizure is specific to the motor cortex. These photomicrographs illustrate that the increase in NMDAR-1 receptor subunit is specifically orientated to the motor cortex.
160
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
creasing ) 400% after a seizure stimulus w14x. This induction of mRNA reaches maximum levels by 30–60 min after the stimulus. Protein levels of NGFI-A are also increased subsequent to a PTZ-induced seizure starting at 1 h post-stimulus and remaining elevated for at least 4 h w13x. These observations suggest that, if NGFI-A Žor a related member of the EGR family. affects NMDAR-1 expression, then a robust increase, such as is seen after a seizure, in these transcription factors would be needed to affect transcription of NMDAR-1. Why would the motor cortex be specifically affected? This phenomenon could be caused by the specific agent employed for seizure induction. PTZ produces a generalized seizure which does not specifically require afferent information to be carried to the somatosensory cortex.
Secondly, it is possible that specific subregions of the neocortex possess an array of mechanisms available to modify the effectiveness of its synapses. For example, the induction of LTP in the cortex appears to be different between motor and sensory cortex w5x. Finally, a second molecular example of motorrsensory differences occurs in the songbird forebrain in response to PTZ. After PTZ, there occurs a dramatically higher increase in NGFI-A ŽZENK. expression in motor areas than in sensory areas w17x. In rat, NMDAR-1 expression appears to be neuronspecific and levels are altered following certain pathological or physiological conditions. For instance, in partially kindled animals, no change in NMDAR-1 mRNA expression was displayed, while after full kindling, NMDAR-1
Fig. 3. Investigation of the abundance of NMDAR-1 mRNA expression through RT-PCR. 2 m g of total RNA extracted from different tissue types after disparate stimulation paradigms were used in RT-PCR experiments. P 33 -labeled nucleotides were added to the PCR reaction to quantify the results. Preliminary experiments demonstrated that the PCR reactions were still in the linear amplification phase. The graph depicts densitometric values of the autoradiographs of the PCR gels. Numbers were normalized to a 100 control value. Results for motor cortex ŽMC. show a trend to increase both 1 and 2 h with the post-2 h increase being significant at the P - 0.005 value Ž n s 7.. After 2 h, the levels of NMDAR-1 mRNA fall but are still slightly above control values. Conclusions from somatosensory cortex ŽSSC. Ž n s 5. and hippocampus ŽHC. at the 2 h Ž n s 5. time post-seizure demonstrate that there are only negligible increases in mRNA expression. The same conclusions were reached for experiments with scintillation counting the PCR gels for interpretation of PCR changes.
P.J. Jensen et al.r Molecular Brain Research 44 (1997) 157–162
161
Acknowledgements This work was supported by National Institute of Health Grant NS 33913 Žto K.J.M... We thank Amy Pletsch for her work in the preliminary stages of this project.
References
Fig. 4. Changes in levels of NMDAR-1 protein after seizure. Western blots were performed on disparate tissue samples to observe any alterations in NMDAR-1 protein levels following a PTZ-induced seizure. Effects from seizure are a significant increase in protein levels both 1 and 2 h post-seizure and significant decreases at 6 and 24 h after stimulation. This gel depicts a time course post-seizure stimulation specifically in the motor cortex. Proteins were detected through the use of Luma-Phos ŽBoehringer-Mannheim. and further analyzed by densitometry using the NIH image software.
mRNA abundance was downregulated specifically in the dentate gyrus granule cells with a maximum decrease 4 h post-stimulation with levels returned to normal within 12 h w20x. A different study, while using the same kindling process, found no change in NMDAR-1 message 24 h after the last stimulation w11x. No change was documented 24 h following both electroconvulsive stimulation ŽECS. w19x or kainate-induced seizure w9x. Stress brings about a 35–45% decrease in NMDAR-1 expression 24 h after induction w3x. Another example is a decrease in NMDAR-1 expression seen in rat spinal cord 7.5 h and 3 days subsequent to unilateral hind paw inflammation w12x. Collectively, these data suggest that NMDAR-1 is regulated in a stimulus-, temporal- and tissue-specific manner. The mechanisms underlying these alterations remain to be elucidated. Preliminary gel-shift experiments from our laboratory demonstrate that NGFI-A, NGFI-C and EGR-3 have the capability of binding to the promoter region of NMDAR-1 w10x. Preliminary co-transfection experiments from our laboratory w10x demonstrate that the EGR family of transcription factors does indeed bring about changes in the expression of a luciferase expression plasmid under the control of the NMDAR-1 5X-flanking region in PC12 cells. The above in vitro results indicate that the EGR family of transcription factors have the potential to affect NMDAR-1 gene expression. The results of this paper shows that increased levels of NMDAR-1 expression occurs at a time when high levels of NGFI-A protein are expected physiologically and therefore may play a role in NMDAR-1 gene regulation.
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