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Changes in mRNA levels for group I metabotropic glutamate receptors following in utero hypoxia–ischemia
Developmental Brain Research 112 Ž1999. 31–37
Research report
Changes in mRNA levels for group I metabotropic glutamate receptors following in utero hypoxia–ischemia Agnes Simonyi, Jian-Ping Zhang, Grace Y. Sun
)
Departments of Biochemistry and Pathology, UniÕersity of Missouri, Columbia, MO 65212, USA Accepted 13 October 1998
Abstract The expression of group I metabotropic glutamate receptors ŽmGluR1 and mGluR5. and inositol 1,4,5-trisphosphate receptor type 1 ŽIP3 R1. mRNA was studied by in situ hybridization in the developing rat hippocampus after in utero hypoxia–ischemia. In utero hypoxia–ischemia was induced by clamping the uterine blood vessels of near-term fetuses for 10 min. Fetuses were delivered surgically, resuscitated and raised by foster mothers until postnatal day 7 and 14. Results indicated a temporal delay in the expression of mGluR1 mRNA in the dentate gyrus of the ischemic animals. The mGluR1 mRNA level was significantly lower in the ischemic animals at postnatal day 7, but reached a similar level as that of controls at postnatal day 14. In utero hypoxia–ischemia did not change the temporal–spatial expression pattern of either mGluR5 or IP3 R1 mRNA in the hippocampus. Between postnatal day 7 and 14, mGluR5 mRNA showed a high and relatively constant expression, whereas IP3 R1 mRNA levels were increased in all regions examined. The differences in the expressions of group I mGluRs indicate that these receptors may have different functions during hippocampal development and may play different roles in excitotoxicity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Metabotropic glutamate receptor 1; Metabotropic glutamate receptor 5; Inositol 1,4,5-trisphosphate receptor type 1; In situ hybridization; In utero hypoxia–ischemia; Hippocampus
1. Introduction Group I metabotropic glutamate receptors ŽmGluR1 and . 5 which are coupled to the phosphoinositide ŽPI. signaling pathway play a crucial role in synaptic plasticity during development, memory formation, and in neurotoxicity including ischemia w29,32x. Activation of these receptors leads to the formation of inositol 1,4,5-trisphosphate ŽIP3 . and diacylglycerol ŽDG. which are involved in the mobilization of Ca2q from intracellular stores and the activation of protein kinase C ŽPKC., respectively. These second messengers modulate a variety of cellular events w5x. In addition, group I mGluRs have other effects including blockade of Kq channels, potentiation of NMDA responses, inhibition or potentiation of AMPA responses, regulation of Ca2q channels, etc. w29,32x.
) Corresponding author. M121 Medical Sciences Building, Biochemistry Department, Columbia, MO 65212, USA. Fax: q1-573-884-4597; E-mail: [email protected]
Immunohistochemical and in situ hybridization studies give evidence of a strong expression of group I mGluRs in the hippocampus in the developing brain w10,33,35x. The distribution pattern of mGluR1 and 5 seems to be complementary. For example, mGluR5 is the predominant receptor in the CA1 area of the hippocampus, whereas mGluR1 is expressed only in interneurons w10x. Moreover, mGluR1 expression gradually increases during postnatal development, whereas mGluR5 level is high at birth and later decreases to adult level w9,10,33x. The developmental peak of quisqualate stimulated PI hydrolysis occurs between postnatal day 6 and 12 in the hippocampus and exhibits a high correlation with periods of intense synaptogenesis w30x. Interestingly, the activation of PI turnover via these glutamate receptors is further increased after a neonatal hypoxic–ischemic insult w11x. One of the known regulatory factors of mGluRs activity during development is the glutamate itself. Glutamate agonists are able to alter the expression of both the mGluR1 and the type 1 IP3 receptor ŽIP3 R1. in developing neurons w6,37x. To study the effect of ischemia on the developmental regulation of the expression of group I mGluRs and IP3 R1
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 2 - 7
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in the hippocampus, we examined mRNA levels by in situ hybridization after in utero hypoxic–ischemic insult in rats at postnatal day 7 and 14. 1
2. Materials and methods 2.1. In utero hypoxia–ischemia All experiments were performed in strict compliance with NIH guidelines and federal regulations. The protocol of this study was reviewed and approved by the University of Missouri-Columbia Animal Care and Use Committee. Timed pregnant Sprague–Dawley rats at 17 and 19 days gestation age were purchased from Sasco ŽOmaha, NE.. The 19-day gestation rats were allowed to deliver normally for use as foster mothers. When the 17-day gestation rats reached 21st day of gestation, they were anesthetized with isoflurane. The uterine horns were exposed and groups of communicating blood vessels branching from the uterine artery and vein were clamped on the right uterine horn for 10 min. The left uterine horn was unclamped and served as control. During the experiment, the fetuses were kept moist and warm by covering with cotton gauze soaked with physiological saline. Both control and hypoxic– ischemic fetuses were delivered surgically and resuscitated. They were raised by the foster mothers until 7 or 14 days. There were no observable differences in behavior or size between the hypoxic and control animals. 2.2. Tissue preparation Rat pups Ž7–8 per group. were decapitated, the brains were removed, frozen in powdered dry ice and stored at y708C. Twelve-micron coronal sections Žthrough the level of the hippocampus. were cut in a Reichert-Jung 2800N cryostat and thaw-mounted onto twice gelatin-coated microscope slides. Thawed sections were briefly dried on a slide warmer at 378C before being stored at y708C until hybridization. 2.3. Probes Oligonucleotides were synthesized on an Applied BioSystems, Model 380B DNA synthesizer and purified by RPC columns according the manufacturer’s instructions ŽDNA Core Facilities, University of Missouri.. The following sequences were used: mGluR1, 45-mer oligonucleotide complementary to bases 1450–1494 w25x; mGluR5, 39-mer oligonucleotide complementary to bases 1640–1678 w1x. The probes used in this study recognize regions common to all published splice variants of mGluR1 and 5. Oligonu-
1
An abstract of this work has been presented ŽNeurosci. Abstr. 22 Ž1995. 1039.
cleotides Ž5 pmol. were 3X end-labeled by incubating with 50 units of terminal deoxynucleotidyl transferase ŽBoehringer-Mannheim, Indianapolis, IN., 50 pmol 35 S-dATP ŽNew England Nuclear, Boston, MA., 200 mM potassium cacodylate, 25 mM Tris–HCl, 0.25 mgrml bovine serum albumin, and 1.5 mM CoCl 2 ŽpH 6.6. for 15 min at 378C. A 1-kb region of the cDNA for IP3 R1 ŽpCMVI-11, kindly provided by Dr. Thomas C. Sudhof, Univ. of Texas ¨ at Dallas. was used to subclone a 943-base pair fragment spanning bases 559-1501 w28x by PCR and inserted into pCReII vector ŽTA Cloning kit, Invitrogen, San Diego, CA.. After linearization with BamHI ŽBoehringer-Mannheim, Indianapolis, IN., the IP3 R1 cRNA probe was generated by in vitro transcription with T7 RNA polymerase. The reaction contained 120 mM ATP, CTP and GTP, and 12 mM 35 S-aUTP ŽNEN, Boston, MA.. 2.4. In situ hybridization At the time of hybridization, slides were warmed to room temperature and loaded into diethylpyrocarbonate ŽDEPC.-treated racks. Sections were fixed in 4% paraformaldehyderphosphate-buffered saline ŽPBS; 0.15 M NaClr1.0 mM KH 2 PO4r6.0 mM Na 2 HPO4 , pH 7.2. for 5 min, rinsed in PBS for 2 min, and soaked in 0.25% acetic anhydride in 0.1 M triethanolamine hydrochlorider 0.9% NaCl ŽpH 8.0. for 10 min. They were rinsed in 2 = SSC Ž1 = SSC; 150 mM NaClr15 mM sodium citrate, pH 7.0., dehydrated through a graded series of ethanol, delipidated in chloroform, rehydrated to 95% ethanol and air dried. Fifty microliters of hybridization buffer was applied to each slide Žtwo sectionsrslide., covered with a parafilm coverslip and incubated at 528C ŽcRNA probe. or at 378C Žoligonucleotide probes. for 20 h in humid chambers. The hybridization buffer for cRNA probe contained 50% formamide, 2 = SSC, transfer RNA Ž250 mgrml., sheared, single-stranded salmon sperm DNA Ž100 mgrml., 1 = Denhardt’s solution Ž0.02% each of BSA, Ficoll and polyvinylpyrrolidone., 10% Žwrv. dextran sulfate ŽMW s 500 000., 100 mM dithiothreitol and 1.0 = 10 6 cpm probe. The hybridization buffer for oligonucleotide probes contained 4 = SSC and 0.75 = 10 6 cpm probe, but all other components were the same. After hybridization, the coverslips were removed in 1 = SSC. Sections Žfor cRNA probe. were washed in 50% formamider2= SSC at 528C twice Ž5 min and 20 min.. Following two 1-min rinses in 2 = SSC at room temperature, the slides were incubated in 2 = SSC containing 100 mgrml RNAse A at 378C for 30 min. The sections were then washed twice for 1 min in 2 = SSC at room temperature. After washing in 2 = SSCr50% formamide at 528C for 5 min, the sections were dehydrated through a graded series of ethanol. Slides Žoligonucleotide probes. were washed four times in 1 = SSCr1 mM DTT Ž15 min each, at 558C., then in 1 = SSCr1 mM DTT at room temperature for 30 min. Slides Žfor both probes. were then dipped in distilled water, immersed in 70% ethanol and air dried.
A. Simonyi et al.r DeÕelopmental Brain Research 112 (1999) 31–37
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Fig. 1. Photomicrograph showing mGluR1 Žtop., mGluR5 Žmiddle. and IP3 R1 Žbottom. mRNA distribution in rat brain coronal sections at postnatal day 7 Žleft. and 14 Žright..
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A. Simonyi et al.r DeÕelopmental Brain Research 112 (1999) 31–37
2.5. Autoradiography and signal quantitation Specific labeling was detected with KODAK BIOMAX MR film ŽEastman KODAK, NY.. For each probe, sections from the four groups were processed identically and exposed for the same length of time. Slides were held against film with standards in X-ray cassettes for two days ŽIP3 R1., five days ŽmGluR5., or seven days ŽmGluR1.. Microdensitometry was performed on the signal over different brain regions using a BioQuant MEG System IV interfaced with a Dage-MTI 70 series video camera equipped with a 2 = NIKON objective mounted onto a bellows system over a light box. Film density was used as an index of the relative level of mRNAs. Standards were used to ensure that the film is not saturated. The hybridization signal was evaluated for each probes as follows. First, the average gray-level was measured for signal and background for each section. Background was subtracted from signal to provide a numerical value of average density over the specified region. This corrected density was averaged over the two brain sections measured for each animal before being evaluated for statistical significance.
3. Results As shown in Fig. 1, prominent expression of mGluR1 mRNA was clearly seen in the dentate gyrus and CA2-4 regions. In contrast, mGluR5 mRNA was abundantly expressed in the CA1 region, as well ŽFig. 1.. Intense labeling was observed in the CA1 region for IP3 R1 mRNA. The signal was very weak in the dentate gyrus at postnatal day 7, but was clearly increased at postnatal day 14 ŽFig. 1.. The distribution patterns of all three mRNAs were consistent with previous reports w10,23,35x.
Fig. 2. Effect of in utero hypoxia–ischemia on IP3 R1 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean"S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and no effect of ischemia, but significant effects of time in CA1 Ž F s132.00, p- 0.0001., CA3 Ž F s 32.69, p- 0.0001. and DG Ž F s 204.69, p- 0.0001..
Fig. 3. Effect of in utero hypoxia–ischemia on mGluR5 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean" S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and no effect of ischemia, but significant effects of time in CA1 Ž F s18.23, p- 0.0003. and in CA3 Ž F s 44.15, p- 0.001..
In utero hypoxia–ischemia did not change the temporal–spatial expression pattern of mGluR5 and IP3 R1 mRNAs in the hippocampus ŽFigs. 2 and 3.. The mRNA level of mGluR5 decreased in CA3 and slightly increased in CA1 between postnatal day 7 and 14 ŽFig. 3.. During the same period of time, IP3 R1 mRNA level dramatically increased in all three regions examined ŽFig. 2.. In utero hypoxia–ischemia caused a temporal delay in the expression of mGluR1 mRNA in the dentate gyrus. Two-way ANOVA revealed a significant interaction between ischemia and time ŽFig. 4.. The mRNA level was significantly lower in the ischemic animals at postnatal day 7, but reached a similar level as that of controls by postnatal day 14 ŽFig. 4..
Fig. 4. Effect of in utero hypoxia–ischemia on mGluR1 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean" S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and effects of either time or ischemia in CA1 and CA3. However, it showed a significant interaction between ischemia and time in the dentate gyrus Ž F s13.70, p- 0.0014.. )Significantly different Ž p- 0.05. from seven-day-old controls ŽC7. using Bonferroni’s t-test. ))Significantly different Ž p- 0.001. from sevenday-old ischemic group ŽI7. using Bonferroni’s t-test.
A. Simonyi et al.r DeÕelopmental Brain Research 112 (1999) 31–37
4. Discussion In utero hypoxia–ischemia is an important cause of perinatal mortality and neurological morbidity. Estimates suggest that between 2 and 4r1000 full-term newborn infants suffer asphyxiation at or before birth, and this type of incidence is greatly magnified and approaches 60% in small premature infants w31,38,39x. After an asphyxial period, some infants recover neurologically intact, whereas others develop permanent deficits. The mechanisms leading to brain injury are still largely unknown, but several factors appear to be involved, e.g., release of excitatory amino acid neurotransmitters, loss of intracellular calcium homeostasis and oxygen free radical formation w15,27x. In turn, these factors may alter signal transduction pathways and, subsequently, gene expression processes. Among the many experimental models, we used a rat model in which perinatal hypoxia–ischemia was induced in utero w22x. This near-term fetal rat model has many advantages, such as low experimental variability, similarity to human pathophysiological situations, parallelism in neurological development between rat and human and suitability for testing possible therapeutic agents by following either acute or long-term changes in brain development. Using this model, Hersey et al. w16x have detected a decrease in carbachol-stimulated phosphoinositide turnover in the cortex two weeks after perinatal hypoxic–ischemic insult. There is evidence that perinatal asphyxia leads to increased incidences of developmental delays, behavior problems and learning disabilities w38,39x. The group I mGluRs have been implicated in developmental plasticity, learning and memory formation, and neuronal injury w27,29,32x. These receptors are shown to be important in the induction of both hippocampal long-term potentiation ŽLTP. and long-term depression ŽLTD. w4,24x. The difference between mGluR1 and mGluR5 is that mGluR1 seems to be critical for hippocampal mossy fiber CA3 LTP, but not for Schaffer collateral CA1 LTP w2,7,21x. Pharmacological studies provided evidence for functional differences between these two receptor subtypes including their response patterns of intracellular Ca2q mobilization, coupling to effector systems, etc. w17,19,20x. Furthermore, their anatomical localizations are different. For example, mGluR1 is found exclusively in neurons, whereas mGluR5 is also localized in glia cells. In the CA1 region, mGluR5 is the predominant receptor, mGluR1 is expressed only in interneurons. In addition, anatomical studies suggested that the PKC limb and not the IP3 one is the more likely route of action for mGluR1 w13x. Our data indicate that the mRNA expression of mGluR1 and mGluR5 in the hippocampus is differentially regulated during early postnatal development and by in utero hypoxia–ischemia. This result suggests that they are likely to play different physiological roles during development. Indeed, a recent study demonstrated that mGluR1 specific
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antagonist was able to reduce the 1S,3 R-ACPD stimulated phosphoinositide turnover only minimally in the hippocampus at postnatal day 9 w9x. This result suggests that mGluR5 is the major mediator of phosphoinositide response to mGluR agonists during the developmental peak in the hippocampus. Unfortunately, studies using whole hippocampi or hippocampal slices would not provide any details on activity in discrete regions. The same is true when Western blot and RT-PCR were used for the analysis of mRNA and protein expression. Although in situ hybridization is less sensitive than RT-PCR, this method can be used to follow changes in discrete hippocampal subfields. Catania et al. w10x and Shigemoto et al. w35x investigated the changes in mRNA expression for mGluRs in rat brain by in situ hybridization during development. Our data are consistent with these reports. In the case of IP3 R1, there is only one publication dealing with postnatal development. Mailleux et al. w23x reported an overall increase in IP3 R1 mRNA level in the brain from G16 until the second postnatal week. Our results are in agreement with their study. Although the CA1 region of the hippocampus is regarded as the most sensitive to hypoxic–ischemic insult, several studies have also demonstrated sensitivity of the neurons in the dentate gyrus to injuries w14,26x. Silverstein et al. w36x examined changes in w3 Hxglutamate binding after perinatal hypoxia–ischemia in seven-day-old rat pups. They found the earliest and most prominent reduction in binding in the dentate gyrus and the changes remained most severe in this area at later times. Furthermore, the postnatal development of the neurons in the dentate gyrus is different from the other parts of the hippocampus. The majority of the granule cells are generated postnatally and synaptogenesis is most active between 4 and 11 days, when the total number of synapses approximately doubles each day w12,34x. Our data showed a significantly lower mRNA level for mGluR1 in the dentate gyrus at seven days after in utero hypoxia–ischemia. Such developmental delay could possibly serve as a homeostatic mechanism, limiting neuronal excitability by restricting the action of glutamate to this type of receptors. In vitro studies showing that the activation of mGluRs can increase NMDA neurotoxicity w3,8x support this hypothesis. Although the activation of group I mGluRs are required for normal development, antagonism of these receptors may exert neuroprotective effect on ischemic insults w27,29x. Interestingly, similar results were demonstrated in adult animals. In a four-vessel occlusion model and a neck-cuff occlusion model of global ischemia, the level of mGluR1 mRNA was significantly decreased in the dentate gyrus, whereas the level of mGluR5 mRNA was unchanged w18x. Our data showed a rapid increase in mGluR1 mRNA level in the dentate gyrus between postnatal day 7 and 14 in ischemic animals. This may indicate an activation of adaptive regulatory mechanisms and a unique role of this receptor during dentate gyrus development.
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Acknowledgements This project was supported in part by the Children’s Miracle Network Telethon Research Fund, University of Missouri.
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Research report
Changes in mRNA levels for group I metabotropic glutamate receptors following in utero hypoxia–ischemia Agnes Simonyi, Jian-Ping Zhang, Grace Y. Sun
)
Departments of Biochemistry and Pathology, UniÕersity of Missouri, Columbia, MO 65212, USA Accepted 13 October 1998
Abstract The expression of group I metabotropic glutamate receptors ŽmGluR1 and mGluR5. and inositol 1,4,5-trisphosphate receptor type 1 ŽIP3 R1. mRNA was studied by in situ hybridization in the developing rat hippocampus after in utero hypoxia–ischemia. In utero hypoxia–ischemia was induced by clamping the uterine blood vessels of near-term fetuses for 10 min. Fetuses were delivered surgically, resuscitated and raised by foster mothers until postnatal day 7 and 14. Results indicated a temporal delay in the expression of mGluR1 mRNA in the dentate gyrus of the ischemic animals. The mGluR1 mRNA level was significantly lower in the ischemic animals at postnatal day 7, but reached a similar level as that of controls at postnatal day 14. In utero hypoxia–ischemia did not change the temporal–spatial expression pattern of either mGluR5 or IP3 R1 mRNA in the hippocampus. Between postnatal day 7 and 14, mGluR5 mRNA showed a high and relatively constant expression, whereas IP3 R1 mRNA levels were increased in all regions examined. The differences in the expressions of group I mGluRs indicate that these receptors may have different functions during hippocampal development and may play different roles in excitotoxicity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Metabotropic glutamate receptor 1; Metabotropic glutamate receptor 5; Inositol 1,4,5-trisphosphate receptor type 1; In situ hybridization; In utero hypoxia–ischemia; Hippocampus
1. Introduction Group I metabotropic glutamate receptors ŽmGluR1 and . 5 which are coupled to the phosphoinositide ŽPI. signaling pathway play a crucial role in synaptic plasticity during development, memory formation, and in neurotoxicity including ischemia w29,32x. Activation of these receptors leads to the formation of inositol 1,4,5-trisphosphate ŽIP3 . and diacylglycerol ŽDG. which are involved in the mobilization of Ca2q from intracellular stores and the activation of protein kinase C ŽPKC., respectively. These second messengers modulate a variety of cellular events w5x. In addition, group I mGluRs have other effects including blockade of Kq channels, potentiation of NMDA responses, inhibition or potentiation of AMPA responses, regulation of Ca2q channels, etc. w29,32x.
) Corresponding author. M121 Medical Sciences Building, Biochemistry Department, Columbia, MO 65212, USA. Fax: q1-573-884-4597; E-mail: [email protected]
Immunohistochemical and in situ hybridization studies give evidence of a strong expression of group I mGluRs in the hippocampus in the developing brain w10,33,35x. The distribution pattern of mGluR1 and 5 seems to be complementary. For example, mGluR5 is the predominant receptor in the CA1 area of the hippocampus, whereas mGluR1 is expressed only in interneurons w10x. Moreover, mGluR1 expression gradually increases during postnatal development, whereas mGluR5 level is high at birth and later decreases to adult level w9,10,33x. The developmental peak of quisqualate stimulated PI hydrolysis occurs between postnatal day 6 and 12 in the hippocampus and exhibits a high correlation with periods of intense synaptogenesis w30x. Interestingly, the activation of PI turnover via these glutamate receptors is further increased after a neonatal hypoxic–ischemic insult w11x. One of the known regulatory factors of mGluRs activity during development is the glutamate itself. Glutamate agonists are able to alter the expression of both the mGluR1 and the type 1 IP3 receptor ŽIP3 R1. in developing neurons w6,37x. To study the effect of ischemia on the developmental regulation of the expression of group I mGluRs and IP3 R1
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 2 - 7
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in the hippocampus, we examined mRNA levels by in situ hybridization after in utero hypoxic–ischemic insult in rats at postnatal day 7 and 14. 1
2. Materials and methods 2.1. In utero hypoxia–ischemia All experiments were performed in strict compliance with NIH guidelines and federal regulations. The protocol of this study was reviewed and approved by the University of Missouri-Columbia Animal Care and Use Committee. Timed pregnant Sprague–Dawley rats at 17 and 19 days gestation age were purchased from Sasco ŽOmaha, NE.. The 19-day gestation rats were allowed to deliver normally for use as foster mothers. When the 17-day gestation rats reached 21st day of gestation, they were anesthetized with isoflurane. The uterine horns were exposed and groups of communicating blood vessels branching from the uterine artery and vein were clamped on the right uterine horn for 10 min. The left uterine horn was unclamped and served as control. During the experiment, the fetuses were kept moist and warm by covering with cotton gauze soaked with physiological saline. Both control and hypoxic– ischemic fetuses were delivered surgically and resuscitated. They were raised by the foster mothers until 7 or 14 days. There were no observable differences in behavior or size between the hypoxic and control animals. 2.2. Tissue preparation Rat pups Ž7–8 per group. were decapitated, the brains were removed, frozen in powdered dry ice and stored at y708C. Twelve-micron coronal sections Žthrough the level of the hippocampus. were cut in a Reichert-Jung 2800N cryostat and thaw-mounted onto twice gelatin-coated microscope slides. Thawed sections were briefly dried on a slide warmer at 378C before being stored at y708C until hybridization. 2.3. Probes Oligonucleotides were synthesized on an Applied BioSystems, Model 380B DNA synthesizer and purified by RPC columns according the manufacturer’s instructions ŽDNA Core Facilities, University of Missouri.. The following sequences were used: mGluR1, 45-mer oligonucleotide complementary to bases 1450–1494 w25x; mGluR5, 39-mer oligonucleotide complementary to bases 1640–1678 w1x. The probes used in this study recognize regions common to all published splice variants of mGluR1 and 5. Oligonu-
1
An abstract of this work has been presented ŽNeurosci. Abstr. 22 Ž1995. 1039.
cleotides Ž5 pmol. were 3X end-labeled by incubating with 50 units of terminal deoxynucleotidyl transferase ŽBoehringer-Mannheim, Indianapolis, IN., 50 pmol 35 S-dATP ŽNew England Nuclear, Boston, MA., 200 mM potassium cacodylate, 25 mM Tris–HCl, 0.25 mgrml bovine serum albumin, and 1.5 mM CoCl 2 ŽpH 6.6. for 15 min at 378C. A 1-kb region of the cDNA for IP3 R1 ŽpCMVI-11, kindly provided by Dr. Thomas C. Sudhof, Univ. of Texas ¨ at Dallas. was used to subclone a 943-base pair fragment spanning bases 559-1501 w28x by PCR and inserted into pCReII vector ŽTA Cloning kit, Invitrogen, San Diego, CA.. After linearization with BamHI ŽBoehringer-Mannheim, Indianapolis, IN., the IP3 R1 cRNA probe was generated by in vitro transcription with T7 RNA polymerase. The reaction contained 120 mM ATP, CTP and GTP, and 12 mM 35 S-aUTP ŽNEN, Boston, MA.. 2.4. In situ hybridization At the time of hybridization, slides were warmed to room temperature and loaded into diethylpyrocarbonate ŽDEPC.-treated racks. Sections were fixed in 4% paraformaldehyderphosphate-buffered saline ŽPBS; 0.15 M NaClr1.0 mM KH 2 PO4r6.0 mM Na 2 HPO4 , pH 7.2. for 5 min, rinsed in PBS for 2 min, and soaked in 0.25% acetic anhydride in 0.1 M triethanolamine hydrochlorider 0.9% NaCl ŽpH 8.0. for 10 min. They were rinsed in 2 = SSC Ž1 = SSC; 150 mM NaClr15 mM sodium citrate, pH 7.0., dehydrated through a graded series of ethanol, delipidated in chloroform, rehydrated to 95% ethanol and air dried. Fifty microliters of hybridization buffer was applied to each slide Žtwo sectionsrslide., covered with a parafilm coverslip and incubated at 528C ŽcRNA probe. or at 378C Žoligonucleotide probes. for 20 h in humid chambers. The hybridization buffer for cRNA probe contained 50% formamide, 2 = SSC, transfer RNA Ž250 mgrml., sheared, single-stranded salmon sperm DNA Ž100 mgrml., 1 = Denhardt’s solution Ž0.02% each of BSA, Ficoll and polyvinylpyrrolidone., 10% Žwrv. dextran sulfate ŽMW s 500 000., 100 mM dithiothreitol and 1.0 = 10 6 cpm probe. The hybridization buffer for oligonucleotide probes contained 4 = SSC and 0.75 = 10 6 cpm probe, but all other components were the same. After hybridization, the coverslips were removed in 1 = SSC. Sections Žfor cRNA probe. were washed in 50% formamider2= SSC at 528C twice Ž5 min and 20 min.. Following two 1-min rinses in 2 = SSC at room temperature, the slides were incubated in 2 = SSC containing 100 mgrml RNAse A at 378C for 30 min. The sections were then washed twice for 1 min in 2 = SSC at room temperature. After washing in 2 = SSCr50% formamide at 528C for 5 min, the sections were dehydrated through a graded series of ethanol. Slides Žoligonucleotide probes. were washed four times in 1 = SSCr1 mM DTT Ž15 min each, at 558C., then in 1 = SSCr1 mM DTT at room temperature for 30 min. Slides Žfor both probes. were then dipped in distilled water, immersed in 70% ethanol and air dried.
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Fig. 1. Photomicrograph showing mGluR1 Žtop., mGluR5 Žmiddle. and IP3 R1 Žbottom. mRNA distribution in rat brain coronal sections at postnatal day 7 Žleft. and 14 Žright..
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A. Simonyi et al.r DeÕelopmental Brain Research 112 (1999) 31–37
2.5. Autoradiography and signal quantitation Specific labeling was detected with KODAK BIOMAX MR film ŽEastman KODAK, NY.. For each probe, sections from the four groups were processed identically and exposed for the same length of time. Slides were held against film with standards in X-ray cassettes for two days ŽIP3 R1., five days ŽmGluR5., or seven days ŽmGluR1.. Microdensitometry was performed on the signal over different brain regions using a BioQuant MEG System IV interfaced with a Dage-MTI 70 series video camera equipped with a 2 = NIKON objective mounted onto a bellows system over a light box. Film density was used as an index of the relative level of mRNAs. Standards were used to ensure that the film is not saturated. The hybridization signal was evaluated for each probes as follows. First, the average gray-level was measured for signal and background for each section. Background was subtracted from signal to provide a numerical value of average density over the specified region. This corrected density was averaged over the two brain sections measured for each animal before being evaluated for statistical significance.
3. Results As shown in Fig. 1, prominent expression of mGluR1 mRNA was clearly seen in the dentate gyrus and CA2-4 regions. In contrast, mGluR5 mRNA was abundantly expressed in the CA1 region, as well ŽFig. 1.. Intense labeling was observed in the CA1 region for IP3 R1 mRNA. The signal was very weak in the dentate gyrus at postnatal day 7, but was clearly increased at postnatal day 14 ŽFig. 1.. The distribution patterns of all three mRNAs were consistent with previous reports w10,23,35x.
Fig. 2. Effect of in utero hypoxia–ischemia on IP3 R1 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean"S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and no effect of ischemia, but significant effects of time in CA1 Ž F s132.00, p- 0.0001., CA3 Ž F s 32.69, p- 0.0001. and DG Ž F s 204.69, p- 0.0001..
Fig. 3. Effect of in utero hypoxia–ischemia on mGluR5 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean" S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and no effect of ischemia, but significant effects of time in CA1 Ž F s18.23, p- 0.0003. and in CA3 Ž F s 44.15, p- 0.001..
In utero hypoxia–ischemia did not change the temporal–spatial expression pattern of mGluR5 and IP3 R1 mRNAs in the hippocampus ŽFigs. 2 and 3.. The mRNA level of mGluR5 decreased in CA3 and slightly increased in CA1 between postnatal day 7 and 14 ŽFig. 3.. During the same period of time, IP3 R1 mRNA level dramatically increased in all three regions examined ŽFig. 2.. In utero hypoxia–ischemia caused a temporal delay in the expression of mGluR1 mRNA in the dentate gyrus. Two-way ANOVA revealed a significant interaction between ischemia and time ŽFig. 4.. The mRNA level was significantly lower in the ischemic animals at postnatal day 7, but reached a similar level as that of controls by postnatal day 14 ŽFig. 4..
Fig. 4. Effect of in utero hypoxia–ischemia on mGluR1 mRNA level in the rat hippocampus at postnatal day 7 and 14. Bars represent mean" S.E.M. of corrected density over the specified regions. Two-way ANOVA revealed no significant interaction and effects of either time or ischemia in CA1 and CA3. However, it showed a significant interaction between ischemia and time in the dentate gyrus Ž F s13.70, p- 0.0014.. )Significantly different Ž p- 0.05. from seven-day-old controls ŽC7. using Bonferroni’s t-test. ))Significantly different Ž p- 0.001. from sevenday-old ischemic group ŽI7. using Bonferroni’s t-test.
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4. Discussion In utero hypoxia–ischemia is an important cause of perinatal mortality and neurological morbidity. Estimates suggest that between 2 and 4r1000 full-term newborn infants suffer asphyxiation at or before birth, and this type of incidence is greatly magnified and approaches 60% in small premature infants w31,38,39x. After an asphyxial period, some infants recover neurologically intact, whereas others develop permanent deficits. The mechanisms leading to brain injury are still largely unknown, but several factors appear to be involved, e.g., release of excitatory amino acid neurotransmitters, loss of intracellular calcium homeostasis and oxygen free radical formation w15,27x. In turn, these factors may alter signal transduction pathways and, subsequently, gene expression processes. Among the many experimental models, we used a rat model in which perinatal hypoxia–ischemia was induced in utero w22x. This near-term fetal rat model has many advantages, such as low experimental variability, similarity to human pathophysiological situations, parallelism in neurological development between rat and human and suitability for testing possible therapeutic agents by following either acute or long-term changes in brain development. Using this model, Hersey et al. w16x have detected a decrease in carbachol-stimulated phosphoinositide turnover in the cortex two weeks after perinatal hypoxic–ischemic insult. There is evidence that perinatal asphyxia leads to increased incidences of developmental delays, behavior problems and learning disabilities w38,39x. The group I mGluRs have been implicated in developmental plasticity, learning and memory formation, and neuronal injury w27,29,32x. These receptors are shown to be important in the induction of both hippocampal long-term potentiation ŽLTP. and long-term depression ŽLTD. w4,24x. The difference between mGluR1 and mGluR5 is that mGluR1 seems to be critical for hippocampal mossy fiber CA3 LTP, but not for Schaffer collateral CA1 LTP w2,7,21x. Pharmacological studies provided evidence for functional differences between these two receptor subtypes including their response patterns of intracellular Ca2q mobilization, coupling to effector systems, etc. w17,19,20x. Furthermore, their anatomical localizations are different. For example, mGluR1 is found exclusively in neurons, whereas mGluR5 is also localized in glia cells. In the CA1 region, mGluR5 is the predominant receptor, mGluR1 is expressed only in interneurons. In addition, anatomical studies suggested that the PKC limb and not the IP3 one is the more likely route of action for mGluR1 w13x. Our data indicate that the mRNA expression of mGluR1 and mGluR5 in the hippocampus is differentially regulated during early postnatal development and by in utero hypoxia–ischemia. This result suggests that they are likely to play different physiological roles during development. Indeed, a recent study demonstrated that mGluR1 specific
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antagonist was able to reduce the 1S,3 R-ACPD stimulated phosphoinositide turnover only minimally in the hippocampus at postnatal day 9 w9x. This result suggests that mGluR5 is the major mediator of phosphoinositide response to mGluR agonists during the developmental peak in the hippocampus. Unfortunately, studies using whole hippocampi or hippocampal slices would not provide any details on activity in discrete regions. The same is true when Western blot and RT-PCR were used for the analysis of mRNA and protein expression. Although in situ hybridization is less sensitive than RT-PCR, this method can be used to follow changes in discrete hippocampal subfields. Catania et al. w10x and Shigemoto et al. w35x investigated the changes in mRNA expression for mGluRs in rat brain by in situ hybridization during development. Our data are consistent with these reports. In the case of IP3 R1, there is only one publication dealing with postnatal development. Mailleux et al. w23x reported an overall increase in IP3 R1 mRNA level in the brain from G16 until the second postnatal week. Our results are in agreement with their study. Although the CA1 region of the hippocampus is regarded as the most sensitive to hypoxic–ischemic insult, several studies have also demonstrated sensitivity of the neurons in the dentate gyrus to injuries w14,26x. Silverstein et al. w36x examined changes in w3 Hxglutamate binding after perinatal hypoxia–ischemia in seven-day-old rat pups. They found the earliest and most prominent reduction in binding in the dentate gyrus and the changes remained most severe in this area at later times. Furthermore, the postnatal development of the neurons in the dentate gyrus is different from the other parts of the hippocampus. The majority of the granule cells are generated postnatally and synaptogenesis is most active between 4 and 11 days, when the total number of synapses approximately doubles each day w12,34x. Our data showed a significantly lower mRNA level for mGluR1 in the dentate gyrus at seven days after in utero hypoxia–ischemia. Such developmental delay could possibly serve as a homeostatic mechanism, limiting neuronal excitability by restricting the action of glutamate to this type of receptors. In vitro studies showing that the activation of mGluRs can increase NMDA neurotoxicity w3,8x support this hypothesis. Although the activation of group I mGluRs are required for normal development, antagonism of these receptors may exert neuroprotective effect on ischemic insults w27,29x. Interestingly, similar results were demonstrated in adult animals. In a four-vessel occlusion model and a neck-cuff occlusion model of global ischemia, the level of mGluR1 mRNA was significantly decreased in the dentate gyrus, whereas the level of mGluR5 mRNA was unchanged w18x. Our data showed a rapid increase in mGluR1 mRNA level in the dentate gyrus between postnatal day 7 and 14 in ischemic animals. This may indicate an activation of adaptive regulatory mechanisms and a unique role of this receptor during dentate gyrus development.
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Acknowledgements This project was supported in part by the Children’s Miracle Network Telethon Research Fund, University of Missouri.
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