Regulation of NMDA receptor subunit mRNA expression in the rat brain during postnatal development

MOLECULAR RESEARCH ELSEVIER

Molecular Brain Research 25 (1994) 209-216

Research Report

Regulation of NMDA receptor subunit mRNA expression in the rat brain during postnatal development M.A. Riva a,b,., F. Tascedda b, R. Molteni b, G. Racagni b a DIBIT, San Raffaele Hospital, Via Olgettina 58, Milan, Italy b Center for Neuropharmacology, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy

Accepted 15 March 1994

Abstract

Different NMDA receptor subunits have been recently cloned. The present paper describes the developmental profile of expression of the NR-1 subunit and three NR-2 subunits (A, B, C) in the rat central nervous system. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of these receptor subunits. We found low levels of NR-1 mRNA (comprising all different splicing isoforms) in newborn rats with a progressive increase of its expression in the following 2-3 weeks. NR-2 subunits can be regarded as 'modulatory' since their expression can produce differences in the properties of NMDA receptors. More than one NR-2 subunits can be expressed in the same brain region. NR-2A and NR-2C are concomitantly expressed in the cerebellum and during development their mRNAs increase with a similar profile from low levels in P-8 rats to maximal expression in P-21 animals. NR-2A and NR-2B are concomitantly expressed in several brain regions with a different ontogenetic profile. In the hippocampus NR-2B mRNA increases rapidly during the first week of life as compared to the NR-2A subunits which at this time is expressed to low levels indicating that NR-2B will probably be dominant in determining the NMDA properties during the first period of life. Our data can provide a molecular correlate with properties of NMDA receptors such as voltage dependent Mg 2÷ block and deactivation kinetics which undergo significant changes during development and have been shown to depend upon the NR-2 subunit co-expressed with the common NR-1 subunit in various brain regions. Key words: NMDA; mRNA; Hippocampus; Ontogenesis; Glutamate; Neurotoxicity; Gene expression

1. Introduction

The N-methyl-D-aspartate ( N M D A ) receptor is a glutamate-gated cation-specific ion channel which plays important roles in many functions of glutamate neurotransmission in the CNS [7]. This receptor subtype is thought to play a key role in synaptic plasticity, including learning and m e m o r y [7]. Moreover, prolonged activation of N M D A receptors can lead to neuronal cell death and might be involved in a n u m b e r of neurodegenerative disorders [5]. Different groups have recently identified by molecular cloning two families of brain N M D A receptors

* Corresponding author. Center for Neuropharmacoiogy, University of Milan, via Balzaretti 9, 20133 Milan, Italy. Fax: (39) 2-20 48 82 11. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00064-L

termed N M D A - R 1 (NR-1) [23] and N M D A - R 2 (NR-2; A, B, C, D) [14,18,21]. The c o m m o n NR-1 subunit occurs in different splice variants with different pharmacological properties [9,10,13,25,28] and expression studies have demonstrated that this homo-oligomeric channel displays many features of native N M D A receptor [23]. On the contrary no detectable currents after the application of glutamate or N M D A can be recorded when only a N R - 2 subunit is expressed [14,18,21]. However co-expression in X e n o p u s oocytes or m a m m a l i a n cells of NR-1 subunits with individual m e m b e r of NR-2 produces an increase by several orders of magnitude in the whole-cell currents activated by glutamate or N M D A [14,18,21]. This is probably indicative of a higher efficency of heteromeric over homomeric channel assembly. It has b e e n therefore suggested that NR-2 subunits can be regarded as 'modulatory' since their expression

2 l I)

M A. Rita et a l / Molecuh*r Brain Rcs'earch 25 (10941 209-216

produces differences in properties such as the voltagedependent Mg 2+ block of the receptor, glycine sensitivity, deactivation kinetics and single channel characteristics [27]. Moreover, as NR-2 subunits have a more restricted pattern of distribution with respect to NR-1 [14,21,23], subunits composition may underlie different properties of NMDA receptors in various brain regions. It becomes therefore interesting the determination of NR-2 mRNA levels in different experimental situations, as their expression can modulate the response of NMDA receptor to glutamate. The NMDA receptor channel complex is crucial for experience dependent synaptic modifications that occur in the developing visual cortex [1]. In young kittens selective NMDA receptor antagonists block experience dependent plasticity and the consolidation of ocular dominance [17]. Moreover, Carmignoto and Vicini have recently demonstrated an activity-dependent decrease in NMDA receptor responses during the development of rat visual cortex [4]. Other changes in the characteristics of the NMDA receptor complex, including increased receptor density, changes in the sensitivity to the voltage dependent Mg 2+ block and to allosteric modulators such as polyamines have been shown to take place during postnatal development [2,16,22,24,29,32]. It has been similarly reported that changes in conductance properties and affinities of other receptor ion channel including nicotinic acetylcholine and glutamate and GABA A receptors take place during development and these changes may result from alteration in subunit composition [3,15,19,20]. Hence, in order to estabilish a molecular correlate between functional properties of the receptor and its gene expression we have investigated the mRNA levels of different NMDA receptor subunits (NR-1, NR-2A, NR-2B and NR-2C) in the rat brain during postnatal development. Our results indicate that a specific spatio-temporal pattern of expression exists for NMDA receptor subunits during postnatal development. During the first week of life, NR-2B subunit will probably be predominant in determining NMDA receptor properties, while the later maturation of NR-2A subunit expression could be indicative of its possible role in modulating the NMDA receptor function in the adult animals. Moreover, the RNase protection we have employed represent a convenient experimental procedure to analyze simultaneously the mRNA levels of four NMDA receptor subunits. The quantitative determination of different receptor subunits in the same biological sample is of considerable utility to analyze and compare their relative expression under basal conditions as well as following pharmacological treatments.

2. Materials and methods Male and female Sprague-Dawley rats of different age (Charles River) were used throughout the experiments. The animals were maintained under a 12 h light/12 h dark cycle with tood a0d v, aler available ad libitum. Rats were sacrificed by decapitation and the brain regions were rapidly dissected, frozen in liquid nitrogen and stored at 7fl°C. 2.1. RNA preparation The tissue from different brain regions was homogenised in 4 M guanidinium isothiocyanate (containing 25 m M sodium citrate pH 7.5, 0.5% sarcosyl and 0.1% 2-mercaptoethanol) and total R N A was isolated by phenol-chloroform extraction according to Chomczynski and Sacchi [6]. Quantification was carrier out by absorption at 2611 nm and R N A was re-precipitated in ethanol for RNase protection assay. In order to verify that equal a m o u n t of total R N A were used in Rnase protection assay, parallel samples were loaded on agarose/formaldehyde gel. run (35 V for 16 h) and stained with ethidium bromide. 2.2. RNA probe preparation The four ptasmids containing the c D N A s for the different N M D A - R subunits were a generous gift of Drs. Peter Seeburg and H a n n a h Monyer [21]. The sequences were arranged in order to obtain proper templates for the in vitro transcription of c R N A probes to be employed in the RNase protection assay. All c R N A probes were generated by a T7 R N A polymerase and [32p]C'TP was used as radiolabelled nucleotide. P F M - N R I , containing a portion of the c D N A for N M D A - R I subunit, was obtained after the removal of a Xhol insert, and consequent ligation, between the c D N A sequence and the polylinker region. This plasmid linearized with BglII was used as a template to yield a 438 b c R N A probe including 24 b of the plasmid polytinker region. P F M - N R 2 A ( N M D A - R 2 A subunit) was obtained after removal of an ApaI insert. This plasmid was linearized with Ncol and used to generate a 187 b c R N A probe, including 16 b of plasmid polylinker region. A HindlIl insert was removed from the original plasmid containing the c D N A sequence for N M D A - R 2 B subunit. Following ligation the plasmid P F M - N R 2 B was obtained. The in vitro transcription of this plasmid linearized with Smal yielded a 310 b c R N A probe, which included 264 b of the receptor subunit sequence and 46 b of the plasmid polylinker region. An analogous procedure was also employed to generate PFMN R 2 C ( N M D A - R 2 C subunit) with removal of a Satl insert from the original plasmid. P F M - N R 2 C was linearuzed with PvulI and used to generate a 244 b c R N A probe, 31 b belonging to the plasmid polylinker region. 2.3. Rnase protection assay The RNase protection assay was performed on a 15-25 ~ g sample of total R N A as described previously [26]: Briefly, after ethanol-precipitation total RNA, obtained from different tissues, was dissolved in 20/,1 of hybridization solution (80% formamide, 40 m M PIPES pH 6.4, 400 m M sodium acetate pH 6.4 and 1 m M E D T A ) containing 150,000 cpm of each 32p-labeled c R N A probes (spec. act. > 10 s c p m / ~ g ) . Four c R N A probes (NR-1, NR-2A, NR-2B and NR-2C) were simultaneously used in the assay. After being heated at 85°C for 10 min, the c R N A probes were allowed to hybridise the endogenous R N A s at 45°C overnight.

M.A. Riva et aL / Molecular Brain Research 25 (1994) 209-216

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At the end of the hybridization, the solution was diluted with 200 /~1 of Rnase digestion buffer (300 mM NaC1, 10 mM Tris HCI pH 7.4, 5 mM EDTA pH 7.4) containing a 1/200 dilution of a RNase cocktail (1 /~g//zl Rnase A and 20 U / ~ I RNase T1) and incubated for 30 rain at 30"C. Proteinase K (10 /~g) and SDS (10 /zl of 20% stock solution) were than added to the sample and the mixture was incubated at 37°C for an additional 15 min. At the end of the incubation the sample was extracted with phenol/chloroform and ethanol precipitated. The pellet, containing the R N A : R N A hybrids, was dried and resusponded in loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 2 ram EDTA), boiled at 95°C for 5 min and separated on a 5% polyacrylamide gel under denaturing conditions (7 M urea). The protected fragments were visualised by autoradiography and their sizes were determined by the use of 32p-end-labelled (T4 polynucleotide kinase) MspI-digested pBR322 fragments.

2.4. RNA calculation The levels of mRNA for different NMDA receptor subunits were calculated by measuring the peak densitometric area of the autoradiography analysed with a LKB laser densitometer. In order to ensure that the autoradiographic bands were in the linear range of intensity different exposure times were used. Data are expressed as % of adult (3 month old) animals. The mean value of the adult within a single experiment was set to 100 and all the other values were expressed as 'percent of adult'. Values of mature animals from different experiments were within 15% of each other.

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2.5. Statistical analysis Statistical evaluation of the changes in mRNA expression of NR subunits during postnatal development was performed using a twoway analysis of variance (ANOVA). Significant changes were determined by Sheffe test (for multiple comparison).

3. Results The RNase protection assay was employed in order to determine simultaneously the m R N A levels for dif-

NR-2A mRNA

Fig. 2. RNase protection assay of NMDA receptor subunits in different brain regions. Twenty #g of total RNA were used in the experiment. Arrows indicate the protected fragments for each receptor subunits. The lane marked as Probes indicates an aliquot (8000 cpm) of the hybridization solution containing the antisense cRNA probes to NR subunits. The X-ray film was exposed for 5 h at - 70°C with intensifying screens.

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ferent NMDA receptor subunits in various brain regions during postnatal development. All cRNA probes were designed in order to avoid any overlapping between probes and protected fragments. Fig. 1 represents a schematic drawing of the probes employed in the RNase protection assay. A low homology region at the 5' end of the D N A sequence

was selected in order to reduce cross hybridization, and consequent backgroung, between different N M D A receptor subunits. NR-1 mRNA detected in our experimets comprises all different isoforms described in the literature [9,13,25,28] as the 438 b cRNA probe used for the RNase protection assay is common to all isoforms.

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M.A. Riva et al. / Molecular Brain Research 25 (1994) 209-216

Fig. 2 shows a typical RNase protection assay for NR subunit mRNA expression in different regions from adult rat brain. The expression of NR-1 is high in cerebellum, hippoeampus, striatum and cerebral cortex with lower levels of expression in hypothalamus and spinal cord. A more restricted pattern of expression is evident for NR-2 subunits, as reported by other authors [18,21,23]. NR-2B is maximally expressed in cerebral cortex, striatum and hippocampus and is not detectable in cerebellum where the highest levels of NR2C mRNA are present. The mRNA for NR-2A is present in various brain areas, the highest expression being measured in the cerebral cortex. We then investigated the pattern of changes of the mRNA levels for these receptor subunits during postnatal development. Fig. 3 shows the developmental profile of expression of NR subunits in rat hippocampus. The expression of NR-1 subunit increased during the first 2 weeks of life, raising from 16% of adult levels observed at P-1 to its maximal expression (118% of adult) measured at P-14. The mRNA levels for this subunit decreased than to mature expression during the following weeks. The developmental profile of expression of NR-2A and NR-2B in hippocampus was quite different between each other. As can be noted in Fig. 3, mRNA levels for NR-2A subunit were low in newborn rats, accounting for only 28% of mature levels in P-8 animals. A large increase in its expression was observed in the second week of life to reach 93% of adult levels in P-14 rats. No significant change of expression was measured thereafter. NR-2B subunit mRNA was already present at 55% of adult levels in P-4 rats, reaching 132% at P-8. Its mRNA expression remained elevated during the following two weeks, with maximal expression at P-14 (149% of adults) and than levelled off to mature levels. Fig. 4 summarizes the pattern of changes of different NR subunits in various brain regions. The expression of NR-1 mRNA increases progressively from P-1 to adult levels: maximal expression is reached during the second or third week of life according to the brain region investigated. To this regard, the developmental regulation in hippocampus is somewhat different as 92% of adult mRNA levels are reached at postnatal day 8 when the expression of NR-1 subunit mRNA elsewhere is still below 50% of mature levels. The maturation of NR-1 expression is slow in the cerebellum as only 50-60% of adult mRNA levels are found on postnatal day 21. The analysis of NR-2A subunit expression indicated that, in several regions, its mRNA is present at very low levels during the first week of life. In fact, as shown in Fig. 3, at P-8 the expression of this receptor subunit in cerebellum, striatum and cerebral cortex was less than 10% of adult levels that were reached after

213

the end of the third week of life. The profile of expression of NR-2A subunit in various brain regions showed a sharp increase between the second and third week of life. A significantly different pattern of expression was found for the subunit NR-2B which, as shown in Fig. 2, predominates in hippocampus and cerebral cortex while it is expressed to very low levels in the cerebellum. The profile of NR-2B expression in the cerebral cortex was quite different from the other subunits. In newborn rats the levels of mRNA were already 50-60% of adult expression and remained constant for the following two weeks. Its mRNA levels increased at P-21, transiently overshooting adult expression. Conversely its developmental profile in the hippocampus indicates that the maximal increase in its expression was observed during the second week of life (Fig. 3) while in striatum a more gradual elevation of its mRNA occurred. NR-2C subunit is expressed predominantly in the cerebellum. However, although its expression is very low in the other brain regions investigated, the use of RNase protection assay allowed us to determine its mRNA levels also in cerebral cortex and striatum. In these two brain areas a steady increase of its gene expression was observed during the first three weeks of life to reach maximal adult levels on P-21. In the cerebellum NR-2C mRNA was not detectable in the first week of life. On P-14 the expression of its mRNA was 35% of adult levels and than increased during the third week of life to reach 85% of maximal expression on P-21 with a pattern which is similar to what observed for NR-2A subunit in the same brain region.

4. Discussion

The present paper describes dynamic changes in the gene expression of four NMDA receptor subunits in the rat brain during postnatal development. We report that in most brain regions the expression for the NMDA receptor subunit mRNA's is very low at birth and increases progressively to reach mature levels around the third week of life. To this regard the pattern of changes in the expression of NR-1 subunit resembles the maturation of different brain regions. In hippocampus maximal mRNA levels are reached within the end of the first week of life, whereas in the cerebellum only 50% of adult expression is reached by postnatal day 21. This pattern of postnatal changes in NR-1 mRNA expression resembles the results of binding experiments during the same period of life. Indeed Morin et al. have shown that in hippocampus and cerebral cortex there is a progressive increase of receptor density, measured by

214

M.A. Rit'a et al. /Moh'cuhtr Braul Research 25 (1994) 209-210

-~H-MK 801, during the first 2 weeks o1' life to reach adult, maximal density around P-21 [22]. NRI subunit exists in at least seven splice variants which arise from alternative splicing. These variants arise from splicing in or out of three alternative exons and most importantly yield receptors with different functional and pharmacological properties [9,10,13,25, 28]. An exon encoding 21 aminoacids can be inserted into the N-terminal domain and this can be combined with different 3' terminal exon deletions. [10,13,25,28] The receptors characterized by N-terminal insertion (NR-Ib) display lower affinity for N M D A and glutamate, lower sensitivity to polyamines and a higher degree of potentiation by protein kinase C activators as compared with the short form (NR-la) [9,10,25]. Expression studies indicate that N R - l a consistently possesses higher agonist potencies and lower antagonist potencies with respect to NR-lb, therefore providing a molecular basis for agonist-preferring and antagonistpreferring N M D A receptor subclasses [13]. Moreover an additional level of diversity for N M D A receptors may result from the combination of NR-I splice variants with NR-2 subunits. As the cRNA probe we used to detect NR1 mRNA does not discriminate between these isoforms, we can not rule out the possibility that specific differences in the expression of such isoforms may take place during postnatal development. There are recent observations to indicate that such changes may indeed take place with in the central nervous system as a higher expression of the isoform lacking the N-terminal insertion was measured at early postnatal stages in hippocampus and cerebellum and not in cerebral cortex [8]. Similarly to our findings Franklin et al. [11] have recently shown that NMDA-R1 gene expression increases progressively from birth to adulthood in different brain regions including frontal cortex, hippocampus and cerebellum. The NR1 subunit possesses many properties characteristic of the N M D A receptor complex although some of these properties are modulated by the NR-2 subunit which is probably co-expressed with NR-1 in native receptors [14,18,21,27]. It was therefore interesting not only to determine the levels of expression for NR-2 subunits but to compare their relative expression in various rat brain region during postnatal development. In the adult rat, NR-2A and NR-2B subunits are concomitantly expressed in various brain regions such as hippocampus, cerebral cortex and striatum (Fig. 2). However, we observed significant differences in their expression profile during postnatal development. In the early postnatal life there is a predominant expression of the NR-2B m R N A as compared to NR-2A. In fact, at postnatal day 8, the m R N A levels for NR-2B in the hippocampus are 132% of adult expression, while only 28% of mature NR-2A m R N A levels are expressed at the same age. A similar pattern was also

found in the cerebral cortex where, in the first week ol life, the expression of NR-2A mRNA was below t0% of adult expression as compared to the levels of NR-2B mRNA ranging from 50 to 70% of mature animals. The predominant expression of NR-2B over NR-2A subunit is more evident if, at different ages, we express their ratio of expression as % of adult values. Theretore, 1 being the ratio NR2B m R N A / N R 2 A mRNA in adult animals, in hippocampus this value is 9.09 at P-4, 4.76 at P-8, decreasing to 1.61 in P-14 and 1.13 in P-21 to indicate again the dominant expression of NR-2B during the first 10 days of postnatal life. Our data indicate that, at least during the first week of life, the subunit NR-2B could determine the properties of NMDA receptor, while in a later phase of postnatal development also NR-2A will come into play to regulate the function of this ionotropic receptor. Beyond changes in NMDA receptor density [22,29] and sensitivity to antagonists [22] and allosteric modulators [32], several experimental observations indicate that functional changes take place during postnatal development and these modifications may be important in neuronal function and plasticity [2,4,12,16,24,30]. Different authors have reported that the sensitivity of the NMDA channel to Mg 2~ block is reduced in newborn rats as compared to adult animals [16,24]. Moreover Carmignoto and Vicini have recently shown kinetic changes in NMDA receptor currents with shorter EPSC in adult as compared to young rats [4]. Many of these properties such as the voltage-dependent Mg 2+ block and decay time constant of the offset current have been shown to depend upon the NR-2 subunit co-expressed with NR-1 subunit. Indeed expression experiments have shown that NR-2A subunit is important in determining the voltage-dependent Mg 2+ block of the NMDA receptor [14,21], Hence the low levels of NR-2A mRNA expression we observed during the first two weeks of life correlates with functional properties of NMDA receptor described by authors who reported the low sensitivity of N M D A receptor to Mg 2+ block in early postnatal life [24]. Further evidences that NMDA receptor properties can be correlated with the expression of specific receptor subunits derive from a recent report showing developmental changes in the sensitivity of N M D A receptor to the novel antagonist ifenprodil [33]. Ifenprodil was shown to displace 125I-MK-801 only with a high-affinity component in newborn rat brain, while from P-7 to mature animal the competition curve was fit to a two-site binding isotherm. The proportion of the low affinity component increases progressively during postnatal days 7-21 resembling the developmental pattern of NR-2A subunit mRNA we observed in our experiments. This observation is confirmed by expression experiments with cloned N M D A receptor subunits indicating that heteromeric N R - I / N R - 2 B receptors have

M.A. Riva et al. / Molecular Brain Research 25 (1994) 209-216

a high sensitivity to ifenprodil while N R - 1 / N R - 2 A receptors display a very low apparent affinity for this drug [33], These data, therefore, provide a good molecular correlate between properties of N M D A receptors during development and expression of different receptor subunits. The larger expression of NR-2B subunit over N R - 2 A in the hippocampus of 1 to 2 week old rats can, therefore, relate to N M D A receptor regulated developmental processes which occur in the hippocampus during this time period. A switch in subunit composition of nicotinic acetylcholine receptors has been shown to be responsible for differences in the gating properties of fetal as compared to adult receptors [19]. Similarly, changes in the levels and distribution of G A B A g and A M P A receptors has been observed during development and may be involved in changes of receptor properties [3,15,20]. These observations suggest that the function of various receptors, including the N M D A complex, can be regulated by the expression of specific receptor subtypes. Our results are in agreement with recent data reported by Watanabe et al. investigating, by in situ hybridization, N M D A - R subunit expression in the mouse brain [31]. Although in the cerebellum of adult rats the expression of NR-2B was barely detectable, we observed a transient elevation of its gene expression between P-8 and P-21 with a maximum around P-14 (data not shown) similar to the findings reported by Watanabe et al. [31]. The observation that in the N M D A receptor different subunits can interact to form heteromeric channel configurations with specific physiological and functional characteristics is indeed very interesting in view of the possible targeting of specific drugs to a certain receptor subunits. As prolonged N M D A receptor activation can lead to neuronal cell death [5], this receptor has become one of the main target for drugs with neuroprotective activity. It is possible that some of these agents may act by modulation of N M D A receptor subunit expression thereby influencing the properties of N M D A receptor channel complex. It is, therefore, important to determine simultaneously the levels of expression of N M D A receptor subunits under different experimental conditions. To this regard the RNase protection assay we have employed and described represent a convenient and reproducible experimental procedure to investigate the gene expression of N M D A receptor subunits.

Acknowledgments We thank Dr. Peter Seeburg and Dr. H a n n a h Monyer for their generous gift of N M D A receptor cDNAs. Partially supported with a grant from Biomed I Concerted Action (PL 921159).

215

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