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

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.

Figure 1. 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 I_NMDA 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 (I_max). At each membrane potential the normalized values (I_norm) 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 K_d value at each potential is plotted as a dotted line. D, the K_d values obtained above were plotted versus the corresponding membrane potential. The dotted lines represent the exemplary voltages from C. The K_d-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).

Figure 2. Postnatal changes in I_NMDA-V relations

Exemplary (panel a) and averaged (means ±s.e.m.; panel b) I_NMDA-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 I_NMDA-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 I_NMDA 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 I_NMDA. The I_NMDA-V relations recorded from cells from the three older groups are almost identical.

Figure 3. Developmental profile of I_NMDA-V relations

Average I_NMDA-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 I_NMDA, 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 I_NMDA upon depolarization. Further changes in voltage dependence are not seen in the P8-P14 (▪) and the P15-adult groups (♦).

Figure 4. Developmental profile of the voltage dependence of NMDA receptors

The average K_d-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 K_d is seen after P4 (P < 0·05; ANOVA). The slope of the K_d-V relations increased after P4 and did not change significantly thereafter.

Figure 5. 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 I_NMDA-V relations are shown from a control cell (A) and from a cell with a severed apical dendrite (B). C, average K_d-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).

Figure 6. Developmental profile of NMDA receptor block by ifenprodil

The I_NMDA 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 I_NMDA in the presence of ifenprodil divided by control I_NMDA is plotted versus postnatal day. The extent of inhibition decreased dramatically after P9, reaching minimal values at about P15.

Figure 7. Effect of ifenprodil on the voltage dependence of NMDA receptors

A, the average K_d-V relations obtained from neurones in the P5-P7 (•), P8-P14 (▾) and > P15 (▪) age groups are shown superimposed on a semilogarithmic scale (means ±s.e.m.). The relations were fitted with a second order linear regression as described in Methods (continuous lines). B, the P5-P7 average K_d-V relation obtained in the presence of ifenprodil (•) was similar to the P1-P4 average K_d-V relation obtained in control saline solution (lower continuous line; P = 0·99, ANOVA), but differed significantly from the P5-P7 average K_d-V relation obtained in control saline solution (upper continuous line; P < 0·05, ANOVA). C, the P8-P14 average K_d-V relations obtained in the presence of ifenprodil (▾) and in control saline solution were identical (continuous line; P = 0·99, ANOVA).

Figure 8. Expression of NR2 subunits in neonate versus adult pyramidal cells

Agarose gel electrophoresis of cDNA amplified from single neonate (A) and adult (B) pyramidal cells. Lane 1 (M) low molecular weight marker Hae III digest of ΦX174. Lane 2, single band 547 bp amplified product of the first PCR for NR2A-C. Lanes 3–5, results of digestion with Bpm I, Bfa I and Sca I, specific for NR2A, NR2B and NR2C, respectively, following purification of the product of the first PCR and a second PCR amplification with the same primers. Lane 6, single band 465 bp amplified product of the first PCR for NR2D. Lane 7 (in A), results of digestion with Xho I following purification of the product of the first PCR and a second PCR amplification with the same primer. Note that in the neonate, NR2B and NR2D were detected (A) whereas in the adult, NR2A and NR2B were detected (B). Numbers to the left of the gels are base pairs (bp).

Figure 9. Developmental changes in expression of NR2 subunits

Single pyramidal cells were harvested for RT-PCR and the expression of each of the four NMDA NR2 subunit mRNA was determined in each cell. The fraction of cells expressing each of the NR2 subunits is plotted as a histogram. The cells were grouped by age into first week (n = 8), third week (n = 10) and fifth week (n = 11). The proportion of cells expressing NR2A mRNA (□, A) increases with age while NR2B mRNA expression (, B) remains relatively constant. NR2D mRNA (, D) expression decreases from 50% in the youngest age group to nil in the oldest age group. NR2C mRNA (▪, C) was detected in one adult cell only.