Portal de Periódicos da CAPES

Early postnatal switch in magnesium sensitivity of NMDA receptors in rat CA1 pyramidal cells

1999; Wiley; Volume: 521; Issue: 1 Linguagem: Inglês

10.1111/j.1469-7793.1999.00099.x

1469-7793

Eilon D. Kirson, Claudia Schirra, Arthur Konnerth, Yoel Yaari,

Magnesium in Health and Disease

The Journal of PhysiologyVolume 521, Issue 1 p. 99-111 Free Access Early postnatal switch in magnesium sensitivity of NMDA receptors in rat CA1 pyramidal cells Eilon D. Kirson, Eilon D. Kirson Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, Jerusalem 91120, IsraelSearch for more papers by this authorClaudia Schirra, Claudia Schirra I. Physiologisches Institut, Universitat des Saarlandes, 66421 Homburg, GermanySearch for more papers by this authorArthur Konnerth, Arthur Konnerth I. Physiologisches Institut, Universitat des Saarlandes, 66421 Homburg, GermanySearch for more papers by this authorYoel Yaari, Corresponding Author Yoel Yaari Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, Jerusalem 91120, IsraelCorresponding author Y. Yaari: Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, PO Box 12272, Jerusalem 91120, Israel. Email: [email protected]Search for more papers by this author Eilon D. Kirson, Eilon D. Kirson Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, Jerusalem 91120, IsraelSearch for more papers by this authorClaudia Schirra, Claudia Schirra I. Physiologisches Institut, Universitat des Saarlandes, 66421 Homburg, GermanySearch for more papers by this authorArthur Konnerth, Arthur Konnerth I. Physiologisches Institut, Universitat des Saarlandes, 66421 Homburg, GermanySearch for more papers by this authorYoel Yaari, Corresponding Author Yoel Yaari Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, Jerusalem 91120, IsraelCorresponding author Y. Yaari: Department of Physiology, Institute of Medical Sciences, The Hebrew University-Hadassah Faculty of Medicine, PO Box 12272, Jerusalem 91120, Israel. Email: [email protected]Search for more papers by this author First published: 07 September 2004 https://doi.org/10.1111/j.1469-7793.1999.00099.xCitations: 75AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract 1 Whole-cell patch-clamp recordings of iontophoretically induced N-methyl-D-aspartate (NMDA) receptor-mediated currents (INMDA) in CA1 pyramidal cells in hippocampal slices from 1- to 40-day-old rats were used to characterize developmental changes in the Mg2+ sensitivity of NMDA receptors. 2 The dose-response relations for extracellular Mg2+ blockade of INMDA indicated a high affinity binding of Mg2+ to NMDA receptors at membrane potentials more negative than −60 mV, independent of postnatal age. 3 Depolarizing the cells unblocked NMDA receptors by decreasing their affinity for Mg2+. The efficacy of depolarization in unblocking NMDA receptors markedly increased after postnatal day 4 (P4), endowing the receptors with a greater voltage dependence. 4 The NR2B subunit-specific NMDA antagonist ifenprodil reduced INMDA in pyramidal cells of all ages. The sensitivity of INMDA to ifenprodil was greatest during the first postnatal week and decreased thereafter, indicating an enhanced contribution of NR2B subunit-containing NMDA receptors to INMDA in the first week after birth. 5 In the first postnatal week, the ifenprodil-insensitive INMDA component had a lower voltage dependence than the total INMDA. In older pyramidal cells, the voltage dependence of the ifenprodil-insensitive component and the total INMDA were similar. 6 In another set of CA1 pyramidal cells, single-cell reverse transcription and polymerase chain reaction (RT-PCR) were used to characterize concomitant developmental changes in NMDA subunit mRNA expression. The mRNA for the NR2D subunit was detected during the first postnatal week in 50% of the cells and disappeared thereafter. The proportion of cells expressing the NR2A and NR2B subunits remained relatively constant throughout the first five postnatal weeks. 7 We conclude that NMDA receptors in hippocampal CA1 pyramidal cells are effectively blocked by Mg2+ at all ages. After 4 days they become much less sensitive to Mg2+ at depolarized membrane potentials. This postnatal switch in voltage control of Mg2+ binding to NMDA receptors may be due to the downregulation of NR2D subunit expression in developing CA1 pyramidal cells. The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a major role in central synaptic transmission (e.g. Armstrong James et al. 1993) and in various forms of neuronal plasticity (Collingridge & Singer, 1990; Constantine Paton et al. 1990; Bliss & Collingridge, 1993). The NMDA receptor is unique among the glutamate receptors in its voltage-dependent blockade by Mg2+ (Mayer et al. 1984; Nowak et al. 1984). It has been suggested that the sensitivity of NMDA receptors to blockade by Mg2+ (and hence to membrane potential) is absent or low at birth and increases thereafter (Bowe & Nadler, 1990; Kleckner & Dingledine, 1991). The resultant enhanced activity of NMDA receptors presumably underlies the increased susceptibility to NMDA receptor-mediated long-term potentiation during the first postnatal weeks (Harris & Teyler, 1984). However, in more recent studies, developmental changes in the Mg2+ sensitivity of NMDA receptors were not observed (Strecker et al. 1994; Khazipov et al. 1995; Kirson & Yaari, 1996). Native NMDA receptors are believed to be multimeric proteins (for review see Mori & Mishina, 1995). Six subunits of the NMDA receptor have been cloned, namely NR1, NR2A-D and NR3A in the rat (Moriyoshi et al. 1991; Das et al. 1998) and the homologous ζ1, ε1–4 and χ1 in the mouse (Kutsuwada et al. 1992; Ciabarra et al. 1995). It has been shown that coexpression in host cells of the NR1/ζ1 subunit with any member of the NR2/ε family will form functional NMDA receptors (Mori & Mishina, 1995). The exact role of the NR3A/χ1 subunit has not yet been defined. However, the four NR1/ζ1-NR2/ε heterodimeric receptors differ in several functional properties, such as Mg2+ sensitivity, offset kinetics of glutamate-induced currents and affinity for different antagonists and modulators (Monyer et al. 1992, 1994; Williams, 1993; Williams et al. 1993). With respect to Mg2+ sensitivity, it was found that recombinant heterodimeric NMDA receptors containing the NR2A or NR2B subunits are more strongly blocked by Mg2+ than those containing the NR2C or NR2D subunits (Monyer et al. 1994; Kuner & Schoepfer, 1996). Given that the expression profile of the different NMDA receptor subunits throughout the brain changes with postnatal development (Mori & Mishina, 1995), it is possible that developmental changes in the Mg2+ sensitivity of NMDA receptors, if they exist, are a consequence of altered NMDA receptor subunit gene expression. Indeed, the developmental shortening of NMDA receptor-mediated excitatory postsynaptic currents has been shown to correlate with the postnatal increase in the fraction of NR2A subunit-containing receptors in both hippocampus (Kirson & Yaari, 1996) and neocortex (Flint et al. 1997). In this study, we characterized quantitatively the postnatal development of NMDA receptor Mg2+ sensitivity. We describe an early postnatal increase in the voltage dependence of Mg2+ binding to NMDA receptors. This switch is paralleled by a decrease in the level of NR2D subunit mRNA expression. Therefore, the expression of NR2D may provide a molecular basis for the low voltage dependence of NMDA receptors early in postnatal development. METHODS Slice preparation Experiments were performed on thin hippocampal slices obtained from 1- to 40-day-old Wistar rats. Methods for preparation of thin slices were similar to those described previously (Edwards et al. 1989; Kirson & Yaari, 1996). Briefly, rats were anaesthetized with ether and decapitated with a guillotine, according to guidelines of The Hebrew University Animal Care and Use Committee. The brain was removed and immediately immersed in ice-cold oxygenated (95% O2-5% CO2) dissection saline solution. The caudal two-thirds of one hemisphere (containing one hippocampus) were glued to the stage of a vibratome (Campden Instruments, UK). Transverse slices, 250 μm thick, were cut from the region of the hemisphere containing the anterior hippocampus. The hippocampal portion was dissected out of each slice and transferred to an incubation chamber containing the oxygenated incubation saline solution at 34°C. After an incubation period of 1 h, slices were transferred, one at a time, to a recording chamber, where they were continuously perfused (2·5 ml min−1) with oxygenated experimental saline solution at room temperature (21–24°C). Solutions The dissection saline solution consisted of (mM): NaCl, 125; KCl, 2·5; NaHCO3, 26·7; Hepes, 13; NaH2PO4, 1·25; glucose, 6·3; CaCl2, 0·5; MgSO4, 4 (pH 7·3). The incubation saline solution was identical except for NaHCO3, which was 22·5 mM (to maintain pH 7·3 at 34°C). The standard experimental saline solution contained (mM): NaCl, 125; KCl, 2·5; NaHCO3, 26·7; CaCl2, 2·5; Hepes, 13; glucose, 12·5 (pH 7·3; osmolarity 300 mosmol l−1). MgCl2 was added at the indicated concentrations. The minimal concentration of MgCl2 tested was 30 μM in order to ensure that micromolar contamination of the solution by Mg2+ from the slice, estimated to be ∼2 μM (Perouansky & Yaari, 1993) would not influence the results. All saline solutions also contained 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 5 μM) to block non-NMDA receptors, tetrodotoxin (TTX; 0·4 μM) to block Na+ currents, CdCl2 (100 μM) to block Ca2+ currents, bicuculline methiodide (10 μM) to block GABA-mediated Cl− currents and glycine (5 μM) to saturate the glycine binding sites in NMDA receptors. The intracellular (pipette) solution consisted of (mM): CsF, 110; tetraethylamonium (TEA), 20; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 10; Hepes, 10; MgCl2, 2; CaCl2, 1 (pH 7·2; osmolarity ∼280 mosmol l−1). All drugs were purchased from Sigma Chemical Co. (Israel) with the exception of CNQX, which was from Tocris Neuramin (UK). Whole-cell recordings Cells in the CA1 field were visualized at ×640 magnification with Nomarski optics using a Zeiss Axioskop microscope. Pyramidal cells were identified by their position in the pyramidal layer, the pyramidal shape of their somata and their prominent apical dendrite. In a few slices, the apical dendritic arbors of the cells were severed using a sharp micropipette. The micropipette was dragged across the slice just apical to the pyramidal cell layer, and the slices were allowed to recuperate for at least an hour. Recording pipettes were pulled from borosillicate glass on a vertical puller (List-medical, Germany) and coated with Sylgard resin (Dow Corning Chemical Co., USA). Pipette resistances were 1–5 MΩ when filled with intracellular solution. After establishing whole-cell recording configuration, series resistance was compensated for by setting the series resistance compensation control of the amplifier (Axopatch 200A) to 70–90%. Experiments in which the series resistance exceeded 20 MΩ were discarded. Iontophoresis A two-barrel electrode was used for NMDA iontophoresis. One barrel (∼100 MΩ) contained 100 mM NMDA and 50 mM NaCl; the second barrel (∼20 MΩ) contained 150 mM NaCl. The tip of the electrode was placed near to the soma of the patched cell. Iontophoretic ejection currents of ∼150 nA were applied for 60 s. A retaining current of up to 10 nA was used to avoid leak of NMDA. Analysis and experimental protocol Currents recorded were filtered on-line at 1 kHz, digitized at a sampling rate of 3 kHz and analysed off-line using a Pentium personal computer and software from Axon Instruments (USA). Analysis was performed on averages from two or three consecutive responses to voltage ramps. The relation between NMDA-induced current (INMDA) and membrane voltage (INMDA-V relation) was obtained for each cell at four different Mg2+ concentrations using the following protocol (Fig. 1). The patched cell was held at an inter-episode membrane potential of −60 mV. The membrane potential was stepped to +30 mV for at least 1 s to allow for maximal inactivation of voltage-gated currents. Subsequently, the holding potential (Vh) was shifted to −100 mV at 0·1 mV ms−1 and then returned to −60 mV. The protocol was first executed in control saline solution, and then repeated after reaching a steady-state response to NMDA iontophoresis (Fig. 1A). The currents recorded in control condition were subtracted from those recorded during NMDA application. The subtracted currents for each Mg2+ concentration were normalized to the maximal outward current at a membrane potential of +30 mV. Finally, the +30 to −100 mV ramp portion of the resulting currents was plotted versus membrane potential (Fig. 1A). Figure 1Open in figure viewerPowerPoint Experimental protocol and analysis A, whole-cell patch-clamp recordings from an exemplary P21 pyramidal cell. The cell was held at +30 mV and stepped to −100 mV with a slow 1 s ramp. This protocol was repeated before and during a steady-state iontophoretic NMDA current. The Mg2+ concentration in the saline solution was 0·03, 0·1, 0·3 or 1 mM Mg2+. B, the control current trace was subtracted from the traces recorded in the presence of NMDA. The resultant INMDA values were then normalized to the peak outward current at +30 mV and plotted versus membrane potential. C, the normalized traces from B were sampled at 30 discrete voltage values. These values were normalized to a linear regression of the area of maximal NMDA conductance (dashed line in B), representing the predicted current values in Mg2+-free saline solution (Imax). At each membrane potential the normalized values (Inorm) were plotted versus the Mg2+ concentration in which they were recorded, and fitted with a simple dose-response curve (see Methods). Exemplary dose-response curves at −64 and −41 mV (▾ and •, respectively) are shown in C. The Kd value at each potential is plotted as a dotted line. D, the Kd values obtained above were plotted versus the corresponding membrane potential. The dotted lines represent the exemplary voltages from C. The Kd-V relation was fitted with both first (between −80 and −30 mV) and second order (over the entire voltage range) linear regressions (continuous lines; see Methods). The INMDA-V relations obtained in each cell were reduced to thirty membrane potential samples each. The INMDA at each potential was then normalized to the predicted maximal current in the presence of 0 mM Mg2+ (Imax). The Imax values were obtained by fitting a linear regression line to the area of maximal NMDA conductance in the measured INMDA-V relations (∼-10 to +30 mV) and extrapolating it to the sampled membrane potentials (Fig. 1A). For each of these potentials, the relation between normalized INMDA (Inorm) and extracellular Mg2+ concentration ([Mg2+]o) was fitted using a simple dose-response equation (Fig. 1A): where Inorm=INMDA/Imax, [Mg2+]o is the extracellular Mg2+ concentration and Kd is the [Mg2+]o of half-maximal inhibition. We used a Hill coefficient of unity, in concordance with a previous study of Mg2+ binding to different recombinant NMDA receptors (Kuner & Schoepfer, 1996), in order to fit Inorm to the four different [Mg2+]o (0·03, 0·1, 0·3 and 1 mM). The fit was constrained to Inorm= 1 (no Mg2+ block) at 0 mM [Mg2+]o. The Kd values thus obtained were plotted against membrane potential on a semi-logarithmic scale for each cell. These Kd-V relations deviated slightly from linearity, as previously described for NMDA receptors expressed in Xenopus oocytes injected with native hippocampal mRNA (Kleckner & Dingledine, 1991) or with recombinant NMDA receptor subunit cRNA (Kuner & Schoepfer, 1996). They were fitted empirically with a second order linear regression (Fig. 1A): where Vh is the membrane holding potential and a, b and c are constants. In order to asses the relative position of the Mg2+ binding site within the transmembrane electric field, the voltage dependence of Mg2+ block of NMDA receptors was quantified by fitting the Woodhull equation (Woodhull, 1973) to the Kd-V relations: where δ is the fraction of the electric field experienced by a Mg2+ ion, z is valency, V is membrane potential, Kd(0mV) is the affinity of the NMDA receptor for Mg2+ at 0 mV and R, T and F have their usual meanings. Because the Woodhull equation assumes linearity, we fitted only the −80 to −30 mV portion of our data where a good approximation of linearity could be assumed and the strength of fit of the Inorm-[Mg2+]o relations was best (r2 > 0·95; see also Kuner & Schoepfer, 1996). Statistics Data are presented as means ±s.e.m. Significant differences between groups of samples were tested with either ANOVA with subsequent post hoc testing, Student's t test, or the Mann-Whitney U test. A significance level of α= 0·05 was used in all tests. Fitting procedures used the Marquardt-Levenberg algorithm to seek parameter values that minimized the sum of the squared differences between the observed and predicted values of the dependent variables. Single-cell RT-PCR In order to characterize the expression profile of NR2 subunit mRNA at different stages of development, pyramidal cell contents were harvested from a separate set of cells as described previously (Plant et al. 1997). Briefly, cells were harvested by applying steady negative pressure to the pipette in the whole-cell recording mode. The flow of the cytoplasm with or without the nucleus into the pipette was monitored visually. In some experiments a larger diameter pipette (2–3 μm) was pressed to the cell surface and the whole soma was aspired without previously obtaining a tight seal. In some of the latter experiments (n= 6), equivalent volumes of extracellular material were harvested from the same slices. No subunit expression was found in these samples, ruling out contamination of the pipette with mRNA from the debris of neighbouring cells. No differences were found between results obtained in the two different harvesting protocols. Under a dissecting microscope, the pipette tip was broken and the contents of the pipette expelled into a PCR tube by applying a positive pressure pulse (N2, 200–400 kPa). The tube contained 2·5 μl of a solution of hexamer random primers (final concentration 5 μM), the four deoxyribonucleotide triphosphates (final concentration 0·5 mM) and dithiothreitol (final concentration 10 mM), to which 20 units of ribonuclease inhibitor and 100 units of Moloney murine leukaemia virus reverse transcriptase (GIBCO-BRL, Eggenstein, Germany) were added (for details see Lambolez et al. 1992; Audinat et al. 1994). The ∼10 μl of reaction mix was incubated at 37°C for 1 h to synthesize single-stranded cDNA, then stored at −80°C until used for PCR amplification. Controls with water and with total RNA, prepared using a guanidium thiocyanate single-step method (Chomczynski & Sacchi, 1987), from the cerebellum of 15-day-old rats were performed in parallel. The PCR amplification of NR2A-D was performed as described by Audinat et al. 1994. To test the same cell for NR2A-C and NR2D, the product of the RT reaction was divided into equal amounts and one half used for the PCR amplification of NR2A-C and the other for NR2D. RESULTS Developmental changes in voltage dependence of NMDA currents To test whether the affinity of NMDA receptors for Mg2+ is developmentally regulated, we compared INMDA values in CA1 pyramidal cells from postnatal day 1 (P1) to postnatal day 40 (P40). Significant differences in INMDA-V relations were observed only in the first postnatal week. Therefore, the results from 37 neurones were grouped as follows: P1-P4, P5-P7, P8-P14 and P15-adult. Exemplary (upper panels) and averaged (lower panels) INMDA-V curves for these four age groups are shown in Fig. 2. It is evident from the INMDA-V curves in the youngest group (Fig. 2A), that NMDA receptors are already highly sensitive to extracellular Mg2+ at birth. Thus at membrane potentials negative to −25 mV, INMDA decreased as [Mg2+]o was elevated. However, the efficacy of depolarization in relieving the Mg2+ block, i.e. its voltage dependence, was much less apparent in P1-P4 than in the older age groups (Fig. 2A–D). Thus, in the neurone shown in Fig. 2Aa, depolarization from −60 to −30 mV in 1 mM Mg2+ caused only a small increase in INMDA, whereas a similar change in older neurones (Fig. 2Ba–Da) caused a greater than 5-fold increase in INMDA. The lesser voltage dependence of Mg2+ block of NMDA currents in the youngest neurones compared to those older than P4 was also apparent in the averaged INMDA-V relations (compare Ab to Bb-Db in Fig. 2). This consistent finding is further illustrated in Fig. 3 by superimposing the averaged INMDA-V relations in 0·1 mM (Fig. 3A) and 1 mM Mg2+ (Fig. 3A). Figure 2Open in figure viewerPowerPoint Postnatal changes in INMDA-V relations Exemplary (panel a) and averaged (means ±s.e.m.; panel b) INMDA-V relations. These relations were recorded from CA1 pyramidal cells from four different age groups: P1-4 (A; n= 5), P5-7 (B; n= 9), P8-14 (C; n= 8) and P15-adult (D; n= 15). In each neurone the INMDA-V relations were recorded in saline solution containing 0·03, 0·1, 0·3 and 1 mM Mg2+. Currents were normalized to the maximal outward current at +30 mV. Note that depolarizing the cell in the presence of > 0·1 mM Mg2+ caused almost no change in the Mg2+ block of INMDA in the youngest age group (A), whereas in the older age groups (B–D) depolarizing the cells from −60 to −20 mV caused a 3- to 5-fold increase in INMDA. The INMDA-V relations recorded from cells from the three older groups are almost identical. Figure 3Open in figure viewerPowerPoint Developmental profile of INMDA-V relations Average INMDA-V relations recorded from CA1 pyramidal cells in the presence of 0·1 mM (A) and 1 mM Mg2+ (B) from the four different age groups. Error bars are omitted for clarity. In the P1-P4 age group (•), depolarizing the cells from −90 to −20 mV caused almost no increase in the INMDA, indicating low voltage dependence of the Mg2+ block. In the P5-P7 groups (▾) an increase in the voltage dependence is seen, leading to a large increase in INMDA upon depolarization. Further changes in voltage dependence are not seen in the P8-P14 (▪) and the P15-adult groups (♦). In order to quantify the developmental changes in the voltage dependence of NMDA receptors, we compared the Kd-V relations obtained for the four age groups studied (Fig. 4). At membrane potentials negative to −60 mV no significant differences in Kd of Mg2+ were found between the different age groups (P > 0·05, Mann-Whitney U test). In all cells tested, Kd was significantly dependent on membrane potential (P < 0·05, ANOVA). The Kd-V relations were nearly linear when plotted on a semi-logarithmic scale, though a better fit was achieved using a second order regression (Fig. 4; see Methods). The Kd-V relation of the first age group was significantly less voltage dependent than those of the older age groups (Fig. 4 and Table 1; P < 0·005, ANOVA). A slight decrease in voltage dependence of Mg2+ blockade was again seen after the second postnatal week (Fig. 4 and Table 1; P < 0·05, ANOVA). Figure 4Open in figure viewerPowerPoint Developmental profile of the voltage dependence of NMDA receptors The average Kd-V relations calculated for all four age groups are shown superimposed on a semilogarithmic scale (means ±s.e.m.; P1-P4, •; P5-P7, ▾; P8-P14, ▪; P15-adult, ♦). The relations were fitted with a second order linear regression as described in Methods (continuous lines). A significant increase in Kd is seen after P4 (P < 0·05; ANOVA). The slope of the Kd-V relations increased after P4 and did not change significantly thereafter. Table 1. Comparison of NMDA receptor sensitivity to Mg2+ during development in the presence and absence of ifenprodil Control 10 μM ifenprodil δ K d(0mV) (mM) δ K d(0mV) (mM) P1–P4 0.65 ± 0.04(5) 1.4 ± 0.2(5) — — P5–P7 1.10 ± 0.04(9) 21 ± 2.2(9) 0.79 ± 0.02(5) 3.6 ± 0.3(5) P8–P14 0.95 ± 0.02(8) 9.3 ± 0.6(8) 0.88 ± 0.04(6) 7.2 ± 0.8(6) P15–adult 0.99 ± 0.02(15) 10.7 ± 0.5(15) 1.1 ± 0.08(8) 21 ± 4.5(8) * Data are presented as means ± S.E.M. The number of cells in each group is shown in parentheses. To estimate the Kd values for Mg2+ block in the absence of an electric field (Kd(0mV)) and the fraction of the electric field felt by the blocking Mg2+ ions (δ), we fitted the linear (-80 to −30 mV) portion of the Kd-V relations with the Woodhull equation and extrapolated it to 0 mV (see Methods; Woodhull, 1973). In the youngest neurones (P1-P4), Kd(0mV) was 1·4 mM and δ was 0·65, whereas in neurones older than P4, Kd(0mV) was greater than 9 mM and δ was ∼1·0 (Fig. 4; Table 1). Taken together, these data suggest that at birth Mg2+ blocks NMDA receptor channels with high affinity but the voltage dependence of this block is low. After P4, the affinity of Mg2+ for the NMDA receptor channels decreases somewhat, whereas its voltage dependence is markedly augmented. Evaluation of space-clamp errors During postnatal development the dendritic arbors of CA1 pyramidal cells increase in both size and complexity (Pokorny & Yamamoto, 1981; Kirson & Yaari, 1996). Even though we applied NMDA to the somata of the cells, age-dependent changes in the quality of space-clamp control could possibly confound our results. In order to evaluate this possibility, we compared data obtained from intact adult pyramidal cells to that obtained from pyramidal cells whose apical dendrites were severed about 50 μm from the soma (see Methods). No significant differences were found in the INMDA-V relations (Fig. 5A and B) and corresponding Kd-V relations (Fig. 5A) obtained from the two groups of pyramidal cells (P= 0·97, ANOVA). Figure 5Open in figure viewerPowerPoint Evaluation of space-clamp control I NMDA-V relations were recorded from adult pyramidal cells at four Mg2+ concentrations (0·03, 0·1, 0·3, 1 mM). In some cells the apical dendrite was severed selectively. Exemplary INMDA-V relations are shown from a control cell (A) and from a cell with a severed apical dendrite (B). C, average Kd-V relations (means ±s.e.m.) from cells with severed apical dendrites (○; n= 4) and from control cells (•; n= 9). No differences in NMDA receptor voltage dependence were seen between the two groups (P= 0·97; ANOVA). Effect of ifenprodil The switch in voltage dependence of Mg2+ block of NMDA receptors may reflect a developmental change in the subunit composition of receptors. If that is the case, then the voltage dependence of the INMDA component that is sensitive to subunit-selective NMDA receptor antagonists may differ from the voltage dependence of the insensitive INMDA component. We tested this notion with ifenprodil, an NMDA receptor antagonist that preferentially blocks NR2B subunit-containing NMDA receptors (Williams, 1993, 1995; Williams et al. 1993; Hess et al. 1998). We reasoned that because the latter receptors are highly voltage dependent (Monyer et al. 1994; Kuner & Schoepfer, 1996), then blocking them selectively with ifenprodil may unmask an INMDA component which is less sensitive to voltage. This would be expected if NR2C or NR2D subunit-containing NMDA receptors contribute substantially to the total INMDA, given that depolarization is less effective in removing the Mg2+ block of these receptors (Kuner & Schoepfer, 1996). Bath application of 10 μM ifenprodil (a concentration which entirely blocks recombinant NR2B subunit-containing NMDA receptors; Williams, 1993; Williams et al. 1993), significantly reduced INMDA values (P < 0·05, t test). This effect was maximal during the first postnatal week and decreased thereafter (Fig. 6). Thus, the magnitude of ifenprodil block of INMDA for the P1-P4, P5-P7, P8-P14 and P15-adult age groups was 85, 84, 53 and 28%, respectively. This age-dependent effect of ifenprodil is consistent with our previous report of a postnatal decrease in the ifenprodil sensitivity of synaptic NMDA receptors in CA1 pyramidal cells (Kirson & Yaari, 1996), presumably consequent to a reduction in hippocampal NR2B mRNA expression after birth (Monyer et al. 1994; Mori & Mishina, 1995; Zhong et al. 1995). Figure 6Open in figure viewerPowerPoint Developmental profile of NMDA receptor block by ifenprodil The INMDA values were recorded in the presence of nominally Mg2+-free saline solution. Ifenprodil (10 μM) blocked NMDA currents significantly (P < 0·05; Mann-Whitney U test) in all cells tested. The average ratio of INMDA in the presence of ifenprodil divided by control INMDA is plotted versus postnatal day. The extent of inhibition decreased dramatically after P9, reaching minimal values at about P15. We then compared INMDA-V relations obtained in control saline solution and in the presence of 10 μM ifenprodil in the age groups P5-P7, P8-P14 and P15-adult (n= 19). This comparison could not be done for P1-P4, because INMDA values obtained at this age were very small (< 50 pA) and, as shown above (Fig. 6), were almost completely blocked by 10 μM ifenprodil. The sensitivity of NMDA receptors to extracellular Mg2+ persisted in the presence of 10 μM ifenprodil (Fig. 7). However, unlike the Kd-V relations in contr