Quantification of AMPAR subunits mRNAs using internal standard cRNA
GluR2 is the key subunit that determines channel properties of AMPARs. To prepare an internal standard for quantification of the amount of GluR2 mRNA, a mutant of GluR2 cDNA was produced by site-directed mutagenesis that exchanged C at position 2098 to G. This substitution abolished the Bsp1286I restriction site specific for GluR2, and created an StuI site (Fig. 1a). cRNA was transcribed in vitro from this mutant cDNA, and designated as R2S cRNA. The transcript was 3.2 kb in length, and comprised the 3′-untranslated region and poly A tail (543 bases). Samples containing either GluR2 or R2S cRNAs alone, or the following GluR2 : R2S ratios, 10 : 1, 10 : 3, 10 : 10, 3 : 10 and 1 : 10, were subjected to RT-PCR using R2 up and AMPA lo primers. The results of restriction analysis of the PCR products are shown in Fig. 1(b), GEL. The product derived from GluR2 cRNA alone was completely digested with Bsp1286I, and that from R2S cRNA alone was completely digested with StuI. The products from mixtures of GluR2 and R2S cRNAs were partially digested with both enzymes. The amounts of both fragments were quantified as the intensity of photo-stimulated luminescence (PSL) determined with an image analyzer (Fig. 1b, PSL). In five different ratios of GluR2 cRNA to R2S cRNA, the relative abundances of GluR2 cRNA were 90.7 ± 0.3%, 77.0 ± 1.5%, 48.8 ± 1.5%, 22.2 ± 1.1%, 9.6 ± 0.7% (mean ± SEM, n = 3), respectively, close to the predicted ratios of 90.9%, 76.9%, 50%, 23.1% and 9.1%. These results indicated that the GluR2-specific competitive RT-PCR maintained original proportions of the GluR2 and R2S cRNA mixture in this 0.1–10-fold range.
Figure 2. Quantification of GluR1–4 mRNAs in forebrain and cerebellar total RNA. (a) The internal standard R2S cRNA was added at various amounts to forebrain or cerebellar total RNA (two different RNA preparations were used for each brain region) prior to GluR2-specific RT-PCR with the upper primer labeled. PSL images are shown. The proportion of Bsp1286I-digested fragment (B lanes) decreased with increases in R2S cRNA input (S lanes: StuI digest, Left B and S lanes: no R2S cRNA added). (b) R2S/GluR2 ratios obtained by PSL were plotted against the number of R2S internal standard cRNA molecules added (circles: cerebellum, squares: forebrain). Each point represents the mean ± SEM (n = 5). The two lines through data plots were drawn according to the best least-squares fit. (c) Total RNA from forebrain or cerebellum was subjected to RT-PCR with AMPA up and AMPA lo primers with the lower primer labeled. PSL image of subunit-specific restriction analysis of PCR products is shown. (d) The graph shows the GluR1–4 proportions obtained after PSL quantification (n = 11 for both forebrain and cerebellum).
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We next examined whether the original proportions of GluR1–4 cRNAs were maintained throughout RT-PCR using simultaneous amplification with AMPA up and AMPA lo primers (Fig. 1a and Lambolez et al. 1992). Equal amounts (108 molecules each) of GluR1–4 cRNAs were mixed and reverse-transcribed, and the RT product was serially diluted and subjected to PCR. PCR products were gel-purified and subjected to a second PCR using the same primers, the lower primer being labeled. The labeled products were digested with subunit-specific restriction enzymes (Fig. 1a) and quantified. As shown in Fig. 1(c), the amplified products contained approximately the same amounts of GluR1–4 even when cDNA was diluted to 10 copies for each subunit (expressed as original cRNA copy number). Upon reduction to 10 molecules, one or two subunits were occasionally undetectable (not shown). However, these drop-outs occurred randomly among GluR1–4 (overall 33% drops-outs regardless of the subunit). These results indicated that the competitiveness is retained even at a very small number of cDNA copies. These results are consistent with those reported previously (Lambolez et al. 1992; Lambolez et al. 1996).
Quantification of AMPAR subunits mRNAs in brain extracts
Using R2S cRNA as an internal standard, we estimated the number of GluR2 mRNA molecules in rat forebrain and cerebellum total RNA. With increases in amount of R2S cRNA added to RNA from either forebrain or cerebellum, the proportion of the fragment derived from R2S gradually increased (Fig. 2a). In Fig. 2(b), the ratios of R2S cRNA relative to GluR2 mRNA (R2S/GluR2) are plotted against the number of R2S cRNA molecules added to each sample. There was a roughly linear relationship between R2S/GluR2 ratio and the number of R2S cRNA molecules in both forebrain and cerebellum samples. From the slopes of the linear regression lines, the amounts of GluR2 mRNAs present in our forebrain and cerebellum RNA samples were estimated to be 9.95 ± 0.18 × 107 molecules/µg forebrain total RNA and 1.61 ± 0.13 × 107 molecules/µg cerebellum total RNA.
Next, the relative amounts of GluR1–4 in rat forebrain and cerebellar total RNA were examined (Fig. 2c,d). GluR1 and GluR2 were the two major components in the AMPAR subunits expressed in the forebrain. On the other hand, GluR1 and GluR4 were the major components found in the cerebellum. The relative abundances of GluR1, GluR2, GluR3 and GluR4 were 33.5 ± 1.4%, 37.9 ± 1.3%, 24.2 ± 1.1% and 6.5 ± 1.3%, respectively, in forebrain RNA (n = 11), and 50.2 ± 2.4%, 13.3 ± 0.6%, 8.4 ± 0.7%, 28.7 ± 1.1% in cerebellar RNA (n = 11). The absolute numbers of GluR1, GluR3 and GluR4 mRNAs were deduced from the above determination of GluR2 mRNA numbers. They were 8.8 × 107 (GluR1), 6.3 × 107 (GluR3) and 1.7 × 107 (GluR4) molecules per µg forebrain RNA, and 6.1 × 107 (GluR1), 1.0 × 107 (GluR3) and 3.5 × 107 (GluR4) molecules per µg cerebellar RNA.
mRNA is generally estimated to represent 3–5% of total RNA. Assuming that it represents 5% of total RNA and that their mean size is 2 kb, the number of mRNA molecules would be 4.6 × 1010 per µg. Therefore, GluR1–4 mRNA would be present at one copy per 170 mRNA molecules in our sample of forebrain RNA and one per 380 in cerebellum.
Quantification of GluR1–4 mRNAs in single hippocampal neurons
We next adapted the quantification of GluR1–4 mRNAs to single-cell mRNAs harvested with patch pipettes. In glia-free cultures (see Materials and methods), the overwhelming majority of cells displayed triangular-shaped somata bearing morphological resemblance to pyramidal neurons. The AMPARs in these cells displayed a linear or outwardly rectifying I–V relation (not shown) indicative of a high GluR2/GluR1–4 ratio.
Typical results of quantification of GluR1–4 mRNAs expressed by a single cultured neuron on day 9 in vitro are shown in Fig. 3(a). After whole-cell patch-clamp recordings, the cell contents were aspirated into the patch-pipette and expelled into a reaction tube. R2S internal standard cRNA (400 molecules) was then added and the mixture was subjected to RT-PCR with AMPA up and AMPA lo primers. The amplified product was gel-purified and second PCR was performed with 32P-labeled upper primers. Quantification of GluR2 mRNA was performed using GluR2-specific PCR (Fig. 3a, left panel). The amplified product was partially digested with either Bsp1286I (B lane) or StuI (S lane). Digestion with both enzymes left no undigested fragment (B + S lane), showing that the amplification was specific for GluR2 and R2S. The amount of Bsp1286I-digested fragment was 2.8-fold higher than that of StuI-digested fragment, indicating that the initial number of GluR2 mRNA molecules was 2.8-fold higher than that of R2S cRNA. Since 400 molecules of R2S cRNA were added as an internal standard, the number of GluR2 mRNAs harvested from the cell was estimated to be 1110.
Figure 3. Quantification of GluR1–4 in single hippocampal neurons in culture. (a) PSL image of restriction analysis of the RT-PCR product obtained from a single neuron after addition of 400 molecules of R2S internal standard cRNA. Left: after GluR2-specific amplification with labeled upper primer, the PCR product was digested with either Bsp1286I (B) or StuI (S). The product was digested to completion by treatment with both enzymes (B + S). NC: no cut, indicates no treatment with these enzymes. The initial amount of GluR2 mRNA was calculated to be 1100 molecules (see text). Right: after GluR1–4 amplification with labeled upper primer, the PCR product was subjected to restriction analysis with BglI (lane 1), Bsp1286I (lane 2), Eco47III (lane 3), EcoRI (lane 4) and StuI (lane 5), which selectively digest GluR1, GluR2, GluR3, GluR4 and R2S, respectively. All: restriction by the five enzymes. NC: no cut. (b) PSL images of RT-PCR and restriction analyses obtained from 3 pyramidal-like neurons from glia-free cultures (left panels) and 2 type-II neurons (right panels). The amounts of R2S internal standard cRNA input are indicated. Note in left panels that the estimate of the number of AMPAR mRNA was little affected by changing the number of R2S cRNA molecules from 400 to 2000 in this neuronal type. These 3 neurons from glia-free cultures predominantly expressed GluR1 and 2. For type-II neurons, the GluR2-specific PCR product was completely digested by StuI (right panels, B and S lanes) with either 400 or 100 R2S cRNA input molecules, indicating that less than 10 GluR2 mRNA molecules were harvested per cell. Type II neurons expressed GluR1 and 4. Lane 5 shows R2S specific digest by StuI. (c) Mean numbers of AMPAR subunit molecules harvested from single neurons (sum is total GluR1–4) in glial-free cultures at day 2 (n = 4, open bars), day 3 (n = 5, stippled bars), day 4–5 (n = 5, double hatched bars) and after day 9 (n = 12, black bars) and single type-II neurons (n = 8, hatched bars).
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Another aliquot of the first PCR product was also subjected to second PCR with AMPA up and AMPA lo primers to determine GluR1–4 proportions. The amplified fragment was completely cut by simultaneous treatment with GluR1, 2, 3, 4 and R2S cDNA-specific enzymes, and proportions of GluR1, 2, 3, 4 and R2S fragments were determined (Fig. 3a, right, lanes 1–5). The ratios of GluR1, 2, 3 and 4 fragments relative to R2S fragment were 2.1, 2.6, 0.7 and 0.3, respectively. Since the number of R2S cRNA molecules was 400, the numbers of GluR1, GluR2, GluR3 and GluR4 mRNAs harvested from the cell were estimated to be 820, 1020, 250 and 130, respectively. The number of GluR2 mRNA molecules estimated here was close to the number obtained from the R2-specific PCR (1110, see above). Indeed, the ratio of the number of GluR2 mRNA molecules estimated using the R2 up and AMPA lo primer pair relative to that estimated using AMPA up and AMPA lo primer pair was 101 ± 8% (n = 15). Thus, the number of GluR2 mRNA molecules could be determined by either method.
As R2S/GluR2 ratios of 0.1–10 were found to be suitable for accurate quantification using PSL, the amount of internal standard should be determined empirically for each neuronal type. We initially added 50 molecules of R2S to harvested pyramidal-like neurons from day 9–16 cultures but failed to detect the fragment from the internal standard R2S cRNA. We next added 20 000 molecules and failed to detect the fragment from GluR2 mRNA. Finally, we found that addition of 400 molecules of R2S was the most suitable for quantification of GluR1–4 mRNAs in this cell type. The results obtained by adding 0, 400 or 2000 molecules of R2S to three different neurons are shown in Fig. 3(b), left panels. In cell 76K1 (upper left), the GluR2-specific PCR yielded a product totally digested by Bsp1286I (lane B) as expected from the absence of R2S cRNA input, and the GluR1–4 PCR showed expression of GluR1 and 2 in this cell (lanes 1,2). Cell 76R5 (middle left, 400 R2S copies), showed predominance of GluR2 over R2S (lanes B, S) and expressed GluR1 and 2. Quantification indicated that 1300 copies of GluR2 and 900 copies of GluR1 mRNA had been harvested from this cell. Material harvested from cell 76R2 (bottom left), which received input of 2000 R2S copies, contained 1400 copies of GluR2 and 470 copies of GluR1 mRNA. Quantitative results obtained in 12 pyramidal-like neurons analyzed after 9–16 days in culture are summarized in Fig. 3(c) (black bars), and showed that the major constituents of AMPARs were GluR1 with values ranging from 185 to 3433 (1150 ± 324 copies, 46% of GluR1–4) and GluR2 with values ranging from 221 to 3465 (1080 ± 273 copies, 43%) in this cell type. The high proportion of GluR2 in GluR1–4 mRNA was consistent with the linear or outwardly rectifying I/V curves of AMPARs expressed by these neurons.
In mixed neuronal and glial hippocampal cultures, a group of non-pyramidal neurons, designated as type-II neurons (Iino et al. 1990; Ozawa et al. 1991), expressed AMPARs characterized by strong inward rectification and high Ca2+ permeability. Consistent with this observation, Bochet et al. (1994) reported that type-II neurons expressed only GluR1 and GluR4, while GluR2 was not detected. However, since the lower limit for detection of AMPAR mRNA was not defined in previous reports, the presence of a small amount of GluR2 mRNA in type-II neurons could not be ruled out. This issue was addressed using the present method.
Hippocampal neurons in mixed neuronal and glial cultures (see Materials and methods) were first characterized electrophysiologically by the I/V relation of their responses to kainate application (see Materials and methods), and after harvesting, the cell's content was analyzed by single-cell RT-PCR with known amounts of internal standard. In eight type-I non-pyramidal neurons with linear or outwardly rectifying I/V relations, analyzed as controls with 400 molecules of R2S, the mean numbers of GluR1, 2, 3 and 4 mRNA molecules per cell were 433 ± 183, 1017 ± 336, 186 ± 81 and 236 ± 67, respectively. The mean total number of GluR1–4 mRNA molecules per cell was 1854 ± 594. The relative abundance of GluR2 to GluR1–4 was 55% (results not shown).
A minority of neurons exhibited kainate responses with strong inward rectification typical of type-II neurons. Quantification of GluR1–4 mRNA in type-II neurons was initially performed using 400 molecules of R2S. Under these conditions, the product of GluR2-specific amplification was completely digested by StuI (Fig. 3b, upper right, lanes B, S). Even when R2S cRNA input was further reduced to 100 molecules, no amplified products derived from GluR2 were detected in any of the eight type-II neurons tested (see example in Fig. 3b, lower right, lanes B, S). As shown in Fig. 3(b), right panels, we detected only GluR1 and GluR4 mRNAs in type-II neurons (except for two cells where the additional presence of GluR3 was detected at 133 and 48 copies per 789 and 494 copies of GluR1–4, respectively). The mean numbers of GluR1 and GluR4 mRNA molecules found in eight type-II neurons were 354 ± 64 with values ranging from 185 to 3433 and 168 ± 36 with values ranging from 39 to 298 (in one cell GluR4 was not detected), respectively (Fig. 3c, hatched bars). The mean number of GluR3 mRNA molecules per type-II neuron derived from the present study was 25 ± 17. If one assumes that type-II neurons are homogeneous in terms of expression of the GluR3 mRNA, our results indicate that below 25 copies per cell, the detection of a given transcript becomes stochastic and reaches the lower limit of the present quantification method (see also Fig. 1c). Therefore, under a similar assumption, the mean number of GluR2 mRNA would be below 25 copies per type-II cell (< 5% of the total AMPAR subunit mRNA, see also discussion).
Developmental changes of AMPAR subunits mRNAs expression in glia-free cultures
Between days 2 and 9, in vitro in glia-free cultures, the size of neurons increased with a marked extension of neurites (Fig. 4a). The membrane capacitance increased from 5.8 ± 0.7 pF (n = 4) on day 2 to 34.8 ± 2.2 pF (n = 12) on day 9 in vitro(Fig. 4b). AMPAR-mediated currents were not detected on day 2 in vitro. Although current responses to 100 µm kainate were scarcely detected in cells on day 3 in vitro, clear responses were seen in all neurons on day 4–5, and their mean amplitude reached 883 ± 114 pA at −60 mV on day 9 in vitro(Fig. 4b). Nevertheless, substantial amounts of AMPAR mRNAs could already be harvested on day 2 and reached maximal levels on day 4–5 in vitro(Fig. 4b). Thus, there was a lag between the expression of AMPAR mRNAs and functional receptors. The results of quantitative analyses of GluR1–4 mRNA harvested from single neurons on day 2 (n = 4, open bars), day 3 (n = 5, stippled bars) and day 4–5 (n = 5, double hatched bars), performed as described above, are shown in Fig. 3(c). The major subunits were GluR1 (46, 37 and 38% of GluR1–4 at day 2, 3 and 4–5, respectively) and GluR2 (25, 22 and 41% of GluR1–4 at day 2, 3 and 4–5, respectively). Both subunits reached their day 9 levels and ratios (see above and black bars in Fig. 3c) at day 4–5, whereas GluR3 and 4 expression decreased between day 4–5 and day 9. The sum of GluR1–4 mRNA harvested reached maximal level at day 4–5 (Fig. 3c).
Figure 4. Developmental changes of AMPAR subunit expression in glia-free cultures. (a) Reverse phase-contrast view of glia-free hippocampal cultures on days 3, 5 and 9 in vitro. Note prominent extension of neurites at day 9. (b) The amount of GluR1–4 mRNA molecules harvested from single cells, the membrane capacitance (Cm) and the amplitude of current responses to 100 µm kainate were normalized to day 9 values and plotted against days in vitro. (c) GluR1–4 were quantified in RNA purified from glia-free culture dishes after cell counting. The number of cells, the amount of total RNA/cell, the amount of GluR2/cell and the amount of GluR1–4/cell were normalized to day 2-values and plotted against days in vitro. (d) The number of GluR2 molecules per cell harvested from single cells (open bars, patch) and that estimated from total RNA in culture dishes (black bars, dish) were plotted against days in vitro. Note the match between the two values until day 4–5 and their separation after day 9.
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AMPAR mRNA quantification results obtained from single cells were next compared with GluR1–4 quantification on RNA purified from glia-free cultures after cell counting (see Materials and methods), performed on 0.1 µg total RNA with 107 copies of R2S internal standard. Although we observed a small decrease in cell number over time, the average amounts of RNA per cell, estimated as the total RNA amount divided by cell number, were 8.3 (100%), 10.2 (123%), 23.2 (280%) and 46.1 pg (555%) per cell on days 2, 3, 4–5 and 9, respectively (Fig. 4c). A steeper increase was observed in the normalized number of GluR2 and GluR1–4 mRNA copies per cell over time. We found 241 (100%), 460 (191%), 1053 (437%) and 3000 copies (1245%) of GluR2 mRNA per cell and 752 (100%), 1288 (171%), 3190 (424%) and 9367 copies (1246%) of GluR1–4 mRNA per cell on day 2, 3, 4–5 and 9, respectively (Fig. 4c). Assuming that mRNA represents 5% of total RNA and that their mean size is 2 kb, the number of mRNA molecules would be 2.3 × 106 per cell at day 9, and GluR1–4 mRNA would be present at one copy per 240 mRNA molecules in our glia-free cultures.
Although GluR2 numbers per cell found here were consistent with numbers found in harvested material from single cells on day 2, 3 and 4–5 (respectively 351, 518 and 1087, see Fig. 4d), we found a discrepancy between these numbers on day 9 (see Fig. 4d, patch and dish bars). This discrepancy was attributed to neurite extension, which is most prominent after day 4–5 (Fig. 4a). Indeed, single cell analysis proceeds mostly with material harvested from somatic patch-clamp. This would explain why both GluR2 and GluR1–4 mRNA copies harvested on single cells reached their maximal numbers on day 4–5 (Fig. 4b), whereas both AMPAR currents and GluR1–4 copies per cell quantified from whole culture extract increased sharply between day 4–5 and day 9 (see Figs 4b and c, respectively). Although this is an indication that a substantial proportion of AMPAR mRNAs are located in neurites, we found no evidence of preferential localization of a given subunit. Indeed, the GluR2/GluR1–4 ratios obtained from single cells, roughly constant between day 4–5 and 9 (41 and 43%, respectively), were also constant in RNA purified from whole cultures (33% and 34%, respectively).