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. 1998 Feb 15;507(Pt 1):13–24. doi: 10.1111/j.1469-7793.1998.013bu.x

Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons

Gabriella Stocca 1, Stefano Vicini 1
PMCID: PMC2230772  PMID: 9490809

Abstract

  1. Pharmacologically isolated miniature NMDA receptor-mediated excitatory postsynaptic currents (mN-EPSCs) were recorded in large visual cortical neurons in layer V of rat cortical slices. Haloperidol (100 μm) and CP101,606 (10 μm), two specific blockers of NMDA receptors comprising NR1/NR2B subunits, were tested on mN-EPSCs in rats at postnatal days 7 and 8 (P7–P8) and P13–P15. At both ages tested, no significant effects of these drugs were seen in the whole population of neurons, although in few neurons at both ages changes in amplitude were observed with haloperidol. Other dopamine receptor antagonists, spiperone and clozapine, failed to decrease mN-EPSCs in cortical neurons at P13–P15.

  2. CP101,606 (10 μm) significantly decreased the amplitude of evoked N-EPSCs (eN-EPSCs) in visual cortical slices from rats at P3–P5, a developmental stage at which mRNA studies have indicated the virtual absence of NR2A mRNA. CP101,606 failed to significantly change evoked AMPA-mediated EPSCs at P5 and eN-EPSCs at P7–P8 and P13–P15.

  3. NMDA receptor-mediated currents were also studied in somatic outside-out patches at P13–P15 with fast application of l-glutamate (1 mm). Haloperidol (50 μm) and CP101,606 (10 μm) blocked these currents in all patches tested. The effect of CP101,606 was concentration dependent.

  4. We suggest that rather early in development synaptic receptors comprising NR1/NR2B subunits could be associated with other subunits so that blockade by haloperidol and CP101,606 is prevented. Moreover, the consistent blockade seen in outside out patches might be ascribed to the confinement of NR1/NR2B receptors to an extrasynaptic population.

Glutamate is the major excitatory neurotransmitter in the CNS (Storm-Mathisen & Ottersen, 1989). Two main classes of glutamate receptors have been identified according to their activation by specific agonists: N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors, which includes those activated by AMPA/kainate (McBain & Mayer, 1994). There is increasing evidence that glutamate receptor activation is crucial for the establishment of functional circuitry during development (Sheetz & Constantine-Paton, 1994), and furthermore NMDA receptors (NR) have been found to play a major role in synaptic plasticity, memory and various diseases which affect the CNS (McBain & Mayer, 1994).

Five subunits of NMDA receptors have been cloned from rat brain, including the NMDA receptor subunit 1 (NR1) and the NMDA receptor subunits NR2A, NR2B, NR2C, and NR2D (McBain & Mayer, 1994; Zukin & Bennet, 1995). The NR1 subunit, occurring in seven different splice variants (Hollmann et al. 1993), is required for NMDA channel function and is ubiquitously expressed in the CNS (McBain & Mayer, 1994). On the other hand, the NR2 subunits (NR2A/B/C/D) confer to the NMDA channel distinct pharmacological and kinetic properties (Monyer, Burnashev, Laurie, Sakmann & Seeburg, 1994; Laurie & Seeburg, 1994; Priestly, Laughton, Myers, Le Bourdelles, Kerby & Whiting, 1995). Although the exact stoichiometry of the native channel is still unknown a pentameric structure has been suggested (Behe, Stern, Wyllie, Nassar, Schoepfer & Colquhoun, 1995).

NR1 and NR2 subunits differ also in the timing of their expression. The NR1 subunit is present throughout the embryonal and postnatal life while the NR2 subunits undergo a diverse temporal and regional distribution during development (Monyer et al. 1994). In situ hybridization studies show that mRNA for the NR2A subunit increases selectively with postnatal development in rats (Monyer et al. 1994). Meanwhile, a parallel decrease in the kinetics of NMDA-mediated excitatory synaptic currents (N-EPSCs) in brain slices has been reported (Carmignoto & Vicini, 1992; Hestrin, 1992; Crair & Malenka, 1995; Flint, Maisch, Weishaupt, Kriegstein & Monyer, 1997). A kinetic change in N-EPSCs has been proposed to underlie the decrease in plasticity occurring during cortical development (Fox, 1995).

Studies performed on cell lines expressing recombinant NR subunits have shown faster deactivation kinetics for receptors comprising NR1/NR2A subunits compared with other combinations (Monyer et al. 1994; Li, Wang, Luo, Wang, Wolfe & Vicini, 1996). At the same time, recent studies have identified pharmacological agents which are able to antagonize in a selective manner NMDARs containing the NR1/NR2B subunits; these include ifenprodil (Williams, 1993), haloperidol (Ilyin, Wittermore, Guastella, Weber & Woodward, 1996) and the novel ifenprodil derivative, CP101,606 (Chenard et al. 1995; Boeckman & Aizenman, 1996).

Recently, antagonism of hippocampal NMDA-mediated synaptic currents by ifenprodil has been shown to be dependent on development (Kirson & Yaari, 1996), implying a developmental decrease in synaptic receptors comprising the NR2B subunit. At the same time, two reports have attempted to correlate the presence of mRNA for NMDA receptor subunits with the decay kinetics of N-EPSCs in neurons in brain slices (Flint et al. 1997; Plant, Schirra, Garashuck, Rossier & Konnerth, 1997). The results of these studies demonstrate that the abundant presence of the NR2A subunit shortens N-EPSC duration in developing hippocampal neurons (Flint et al. 1997) but it is not sufficient to produce fast deactivation kinetics in medial septal neurons (Plant et al. 1997). In order to characterize further the still unknown subunit composition of the native NRs in excitatory synapses of cortical neurons, we tested the effect of NR1/NR2B-selective antagonists on spontaneous and evoked N-EPSCs in visual cortical neurons from brain slices during postnatal development. For comparison, we also studied properties of NMDA receptors in outside-out patches excised from the soma of these neurons where excitatory synapses are mostly absent (Peters, 1985).

METHODS

Brain slice preparation

Slices from the occipital neocortex (200 μm) were obtained from Sprague-Dawley rats at postnatal days 3-15 (P3-P15) decapitated with scissors while under ether anaesthesia. The slices were allowed to recover for about 1 h at 37°C in an oxygenated extracellular medium (composition described below) containing 2 mM MgCl2, and then transferred to the recording chamber. In animals older than P7, cells in layer V were visually identified using an upright microscope equipped with differential interferential contrast (DIC) Nomarski optics (UEM, Zeiss, Germany) and water immersion × 40 objective lens with a long working distance (2 mm). In younger rats (P3-P5), cells in layers underlying the cortical plate were investigated (Kim, Fox & Connors, 1995).

Electrophysiological recording methods

Cortical neurons were voltage clamped at -60 mV in the whole-cell recording configuration using the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981), at room temperature (21-23°C). The resting potential of the neurons was briefly assessed under current clamp and ranged between -55 and -65 mV. The recording chamber was continuously perfused at 5 ml min−1 with a nominally Mg2+-free oxygenated extracellular medium composed of (mM): NaCl, 120; KCl, 3.1; K2HPO4, 1.25; NaHCO3, 26; CaCl2, 2.0; glucose, 1.25; and glycine, 1-10 μM. The solution was maintained at pH 7.4 by bubbling with 5 % CO2+ 95 % O2. NMDA receptor-mediated synaptic responses were pharmacologically isolated by picrotoxin (15 μM; Sigma), and 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX; 5 μM, Tocris). Synaptic activity was stimulated by including 4-aminopyridine (4-AP, 2 mM; Sigma). When indicated, 3-[(±)-2-carboxypiperazin-4-yl]-propy-1-phosphonic acid, (CPP, 10 μM; Tocris) and tetrodotoxin (TTX, 1 μM; Sigma) were also included. (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP101,606; Chenard et al. 1995) was a gift from Dr Richard Woodward (Acea Pharmaceuticals, Irvine, CA, USA). Clozapine (Research Biochemicals Inc. (RBI), Natick, MS, USA), haloperidol (RBI), spiperone (RBI) and CP101,606 were diluted in dimethylsulphoxide (DMSO, < 0.1 % final concentration). All drugs were superfused through parallel inputs to the perfusion chamber. We have no reason to believe that penetration of the slices by CP101,606 and haloperidol was poor because perfusion with another NMDA receptor antagonist (i.e. CPP) very rapidly and fully antagonized N-EPSCs.

Electrodes were pulled in two stages on a vertical pipette puller from borosilicate glass capillaries (Wiretrol II, Drummond, Broomall, PA, USA). Typical pipette resistance was 4-10 MΩ. Intracellular (patch pipette) solutions contained (mM): potassium gluconate, 145; EGTA, 5; MgCl2, 5; ATP-Na, 5.0; GTP-Na, 0.2; and Hepes, 10; adjusted to pH 7.2 with KOH. In some recordings caesium methanesulphonate (145 mM) was substituted for potassium gluconate in which case the pH was adjusted with CsOH. Whole-cell recordings were performed with a patch-clamp amplifier (Axopatch-1D; Axon Instruments). After pipette capacitance compensation in the cell-attached mode, series resistance was assessed in whole-cell recording mode from the instantaneous capacitive current in response to short test pulses delivered once every minute of recording time, and 50-70 % compensated. We accepted only cells with less than 15 MΩ series resistance and we discarded cells where a change greater than 15 % was observed.

Electrically evoked currents were elicited using a bipolar electrode made of two tungsten wires (A-M System, Inc., Everett, WA, USA) insulated except at their tips. The distance between the two tips was approximately 100 μm to favour a focused and monosynaptic stimulation in the vicinity of the neuron from which the recording was taken. The EPSCs were probably derived from excitatory afferents from subcortical structures, as well as from intrinsic cortical connections. The electrical stimuli (square-wave electric pulses of 100-800 μA in intensity and 200 μs in duration) were delivered at about 0.14 Hz. Stimulation in younger rats (< P7) required stronger intensity stimulation. Stimulus intensity and duration were adjusted to obtain varying degrees of maximal response as needed, and then maintained constant throughout the recording.

Fast application technique

For fast application of L-glutamate, we used a computer-driven piezoelectric translator (PZ 150M; Burleigh Instruments, Inc., Fishers, NY, USA) to which a double-barrelled theta tubing was attached. Outside-out membrane patches were excised from the neurons just after establishing the whole-cell configuration and positioned in front of the double-barrelled applicator. In each barrel we used control solution with NBQX (5 μM) with and without L-glutamate (1 mM; flow rate, 0.25 ml min−1). After each recording, on- and off-rates, as well as pulse duration, were measured by ‘blowing off’ the patch and recording currents generated by the liquid junction potential due to a 50:1 dilution of the L-glutamate-containing solution (Lester & Jahr, 1992; Colquhoun, Jonas & Sakmann, 1992). On- and off-rates were typically less than 0.2 ms, and 1 ms was the minimal duration of the liquid junction currents measured. For fast application of L-glutamate with haloperidol and CP101,606, we rapidly exchanged the solutions in both barrels by means of solenoid valves connected to a vacuum. Haloperidol (50 μM) and CP101,606 at increasing concentrations (0.1-10 μM) were added to both control and L-glutamate-containing solutions.

Data collection and analysis

Currents were filtered at 1 kHz with an 8-pole low-pass Bessel filter (Frequency Devices), digitized at 2.5-4 kHz using an IBM-compatible microcomputer equipped with Digidata 1200 data acquisition board (Axon Instruments) and pCLAMP 6.03 software (Axon Instruments). Off-line data analysis, curve fitting, and figure preparation were performed with pCLAMP 6.03 and Origin (Microcal, Northampton, MA, USA) software. Spontaneous miniature N-EPSCs were recorded in the presence of TTX (1 μM) and analysed with dedicated software, provided by Dr Stephen Traynelis (Emory University, Atlanta, GA, USA). Peak amplitudes were measured at the absolute maximum of the currents, taking into account the noise of the baseline (typically 1.3 pA) and noise around the peak. Synaptic currents were identified as downward deflections greater than 3 times the s.d. of the baseline noise. Events with slow onset kinetics (> 30 ms) were discarded as possibly being due to the summation of multiple individual events occurring without synchronization and amplitude was not measured from overlapping events. Due to the limited frequency of occurrence, at least fifty events were measured in each cell. Fitting of decay times of the averaged evoked glutamate-activated NMDA receptor-mediated currents (NMDACs) recorded from excised patches and of evoked N-EPSCs (eN-EPSCs) was performed using a simplex algorithm least-squares exponential fitting routine. Double exponential equations of the form:

graphic file with name tjp0507-0013-mu1.jpg

where If and Is are the amplitudes of the fast and slow decay components, and τf and τs are their respective decay time constants, were used to fit the data from the current peak to the baseline. To compare decay times between different experimental conditions we used a weighted mean decay time constant:

graphic file with name tjp0507-0013-mu2.jpg

Events lists and single-channel current open time histograms were prepared using the pCLAMP 6.03 software suite. Subconductance and multiple opening levels were excluded from the dwell time analysis. Data values are expressed as means ±s.e.m. and P values represent the results of Student's paired t tests, with prior analysis of variance unless otherwise indicated.

RESULTS

NMDA-mediated EPSCs in cortical slices

Large pyramidal neurons in layer V of visual cortical slices from young rats at P7-P8 (n= 10) and P13-P15 (n= 23) were investigated after visual identification. Following the formation of a gigaseal, the cells were voltage clamped at -60 mV in the whole-cell recording configuration and spontaneously occurring, glutamatergic EPSCs could be observed. In order to isolate miniature NMDA-mediated EPSCs (mN-EPSCs), the experiments were performed in the presence of NBQX (5 μM), picrotoxin (15 μM), 4-AP (2 mM) and TTX (1 μM) in a nominally Mg2+-free solution, to relieve the magnesium block of NMDA channels. Under these experimental conditions, small spontaneous inward currents could be detected (Fig. 1, upper traces), due to presynaptic glutamate release activating NMDA receptors as indicated by their blockade with 10 μM CPP (n= 10; not shown). When mN-EPSC amplitudes were compared in visual cortical neurons in the two age groups, the mean amplitudes were significantly larger at P13-P15 (18.8 ± 1.4 pA) than at P7-P8 (8.0 ± 0.8 pA, P < 0.01, independent t test). In the majority of neurons from rats at P7-P8, no spontaneous N-EPSCs were observed after TTX application. In the neurons where mN-EPSCs were observed, their frequency was not statistically different (0.13 ± 0.02 Hz) from that measured in neurons at P13-P15 (0.22 ± 0.03 Hz).

Figure 1. Effects of haloperidol and CP101,606 on mN-EPSCs in developing cortical neurons.

Figure 1

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A, selected sweeps illustrating mN-EPSCs in a visual cortical cell before (upper 4 traces), and during bath application with 100 μM haloperidol (lower 4 traces). The mean mN-EPSC amplitudes were 15.5 ± 7.2 pA in control and 15.4 ± 5.7 pA with haloperidol (mean ±s.d., n= 164). B, mN-EPSCs in visual cortical neuron before (upper 4 traces) and during CP101,606 application (lower 4 traces). In this neuron, peak mN-EPSC amplitudes were 16.1 ± 9.2 pA before and 17.5 ± 5.7 pA after the drug (mean ±s.d., n= 113). C, mN-EPSCs in a neuron in a visual cortical slice from a P7 rat. CP101,606 (10 μM, lower 4 traces) failed to significantly alter mN-EPSC amplitudes which were 4.6 ± 1.2 pA before and 4.2 ± 1.7 pA after the drug (mean ±s.d., n= 85). D, a summary of the effects exerted by haloperidol (Halo), clozapine (Cloza) and spiperone (Spip) (all at 50 μM) on the averaged peak amplitude of mN-EPSCs in visual cortical neurons at P13-P15. The asterisk indicates a statistically significant variation (paired t test, P < 0.05). ▪, control; Inline graphic, Halo, Cloza, Spip. E, a comparison of the effects of 10 μM CP101,606 on the averaged peak amplitude of mN-EPSCs in visual cortical neurons at P13-P15 and at P7-P8. ▪, control; Inline graphic, CP101,606. No statistically significant variations were observed. F, a comparison of the effects of 10 μM CP101,606 on the frequency of occurrence of mN-EPSCs in visual cortical neurons at P13-P15 and at P7-P8. ▪, control; Inline graphic, CP101,606. The asterisk indicates a statistically significant variation (paired t test, P < 0.05).

Haloperidol and CP101,606 fail to inhibit mN-EPSCs in cortical slices

We investigated the effects of haloperidol and CP101,606 on mN-EPSCs. For these studies, a maximal concentration of haloperidol and CP101,606 were used to selectively block NMDA responses mediated by recombinant receptors containing NR1/NR2B subunits but not receptors formed by combinations of the NR1/NR2A subunits (Ilyin et al. 1996; Boeckman & Aizenman, 1996). At P13-P15, mN-EPSCs were measured before and after delivering 100 μM haloperidol (n= 19) and 10 μM CP101,606 (n= 6). Figure 1A and B shows examples of the lack of antagonism by haloperidol and CP101,606. In Fig. 1C, mN-EPSCs are shown from one of the eight neurons at P7-P8 where CP101,606 (10 μM) was tested and also found to have little effect.

At P13-P15, haloperidol significantly decreased the mean peak amplitude of the mN-EPSCs in one of the neurons studied while it produced a significant increase in three additional neurons. In cells at this age, as well as in the population of cells at P7-P8, CP101,606 failed to decrease significantly the mean peak amplitude of the mN-EPSCs in all cells tested. We performed statistical tests to assess the significance of variation in the mean peak current amplitude following administration of haloperidol and CP101,606 in neurons at P13-P15 and P7-P8, and the results of this analysis, illustrated in Fig. 1D and E, show a lack of effect of these drugs on the amplitude of mN-EPSCs when the cell population was considered globally.

In different neurons of the visual cortex, haloperidol and CP101,606 produced both increases and decreases in the frequency of occurrence of mN-EPSCs. Mean values are illustrated in Fig. 1F. The variation of frequency induced by the CP101,606 was significant only for neurons at P7-P8 (-25 ± 8 %), while it was not significantly different in any neurons at P13-P15 where haloperidol and CP101,606 were tested (-12 ± 44 and -2 ± 6 %, respectively). At all ages, with both haloperidol or CP101,606 regression analysis revealed no significant correlation between the variations in mN-EPSC amplitude and frequency in the cells studied.

We also tested, for comparison with haloperidol, the effect of other dopamine receptor antagonists (Goodman & Gilman, 1990). Our unpublished results indicated that both spiperone and clozapine, at concentrations of 50 μM, did not antagonize recombinant NR1/NR2B receptors in mammalian transfected cells. The results shown in Fig. 1D derived from eight visual cortical neurons at P13-P15 indicate that clozapine failed to alter mean mN-EPSC peak amplitude while spiperone produced a slight but significant increase of this parameter.

Pharmacological antagonism of evoked N-EPSCs

Since we failed to observe a consistent blockade of mN-EPSCs by either CP101,606 or haloperidol in visual cortical neurons as early as P7-P8, we studied the NMDA receptors at synapses in younger rats, as early as P3. To investigate the nature of the native receptor composition at this developmental stage when synaptic connections are barely established, we had to electrically stimulate N-EPSCs (eN-EPSCs). This approach, however, raised the concern that blockade of voltage-gated Ca2+ conductances by NR1/NR2B antagonists (Fletcher, Church & MacDonald, 1994; Church, Fletcher, Baxter & MacDonald, 1994) could combine with the postsynaptic antagonism of NR1/NR2B receptors. Since the profile of action of CP101,606 on voltage-gated Ca2+ conductances has not been established, we tested this compound on AMPA-mediated EPSCs (eA-EPSCs) evoked in visual cortex slices in order to investigate possible presynaptic modulation. As illustrated in Fig. 2A and C, CP101,606 (10 μM) did not change the peak amplitude of eA-EPSCs (n= 7 neurons at P5, variation -4 ± 7 %; n= 7 neurons at P14, variation -7 ± 5 %). In Fig. 2B is shown an example of the reduction in eN-EPSCs induced by CP101,606 in a neuron at P5, and a summary of the results obtained in eighteen visual cortical neurons at P3-P5 is shown in Fig. 2E. At P3-P5, CP101,606 significantly decreased the mean peak amplitude of the eN-EPSCs in all but two of the neurons studied. CP101,606 was also tested in nine visual cortical neurons at P7-P8 and in eleven neurons at P13-15 (Fig. 2E; one example in Fig. 2D). The mean peak amplitude of the eN-EPSCs was significantly decreased in four neurons at P7-P8 and two neurons at P13-P15. Within each age group considered, CP101,606 reduced significantly the amplitude of eN-EPSCs only in the P3-P5 neuronal population (Fig. 2E). In P3-P5 neurons, we were not able to carry on with the whole-cell recording for a long time and even a 20 min wash was not sufficient to obtain a full recovery from the effect of CP101,606 at the concentration used. The extent of recovery at this time was 75 ± 12 %. The decay phase of eN-EPSCs was best described by double exponential curves at all ages tested. To facilitate comparison between age groups we used a time constant (τw) that is the weighted mean of the fast and slow time constants. Mean values for τw were similar at P3-P5 (418 ± 40 ms) and at P7-P8 (440 ± 46 ms) but τw was significantly faster at P13-P15 (155 ± 24 ms, P < 0.01, independent t test). In Fig. 2F, the reduction in the amplitude of eN-EPSCs produced by 10 μM CP101,606 in neurons at different ages is reported as a function of the τw values measured in each neuron studied. No significant correlation between the two parameters was observed within each age group, but when data from different ages were pooled together a significant correlation coefficient (r) of 0.35 was observed (P < 0.05), indicating that slower currents in younger animals were more prone to reduction by CP101,606. The time constants and relative proportion of the fast component derived from the double exponential fitting (%F) of the eN-EPSCs at P3-P5 (τf= 121 ± 17 ms, τs= 637 ± 69 ms, %F= 34± 5), at P7-P8 (τf= 73 ± 14 ms, τs= 562 ± 44 ms, %F = 28 ± 8) and at P13-P15 (τf= 62 ± 8 ms, τs= 494 ± 126 ms, %F= 61± 9) were not significantly different before and after perfusion with 10 μM CP101,606.

Figure 2. CP101,606 blockade of evoked EPSCs in cortical neurons.

Figure 2

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A and C, average AMPA-mediated EPSCs were recorded in the presence of CPP (10 μM) and picrotoxin (15 μM) and were obtained from at least ten consecutive events recorded at a holding potential of -60 mV from two visual cortical neurons at P5 (A) and at P14 (C). EPSCs were evoked by stimuli consisting of pulses (200 μs; 100-800 μA at 0.14 Hz) applied through a bipolar tungsten electrode. CP101,606 (10 μM) failed to alter the average eA-EPSCs at both ages. B and D, average evoked NMDA-mediated EPSCs were recorded in the presence of NBQX (5 μM) and picrotoxin (15 μM) and were obtained from at least ten consecutive events recorded at a holding potential of -60 mV from two visual cortical neurons at P5 (B) and P14 (D). CP101,606 selectively decreased the mean eN-EPSC amplitude at P5 but not at P14. The double exponential curves that best describe the eN-EPSC decay are shown with their respective time constant values (τf, and τs), the weighted mean time constants (τw) and the relative proportion of the fast component (%F). The individual exponential curves are also shown (dotted lines). E, a comparison of the effects of 10 μM CP101,606 on the peak amplitude of averaged eN-EPSCs in visual cortical neurons at different age groups. The asterisk indicates a statistically significant variation (paired t test, P < 0.05). F, scatter plots at different age groups of the percentage reduction in the mean amplitude of eN-EPSCs produced by bath perfusion with 10 μM CP101,606 as a function of the time constant (τw) deriving from the weighted mean of the fast and the slow time constants of a double exponential fitting of the decay of control eN-EPSCs.

NMDA receptor currents in excised somatic outside-out patches

We also compared N-EPSCs generated by activation of synaptic NMDA receptors in response to currents produced by the rapid activation of somatic NMDA receptors in twenty-seven visual cortical neurons at P13-P15. Brief (1 ms) applications of L-glutamate (1 mM) with a piezoelectric translator evoked inward currents (NMDACs) in excised patches held at a holding potential of -60 mV, (peak amplitude range, 4-301 pA; mean, 71 ± 11 pA). Four selected individual examples of such currents recorded from a neuron at P14 are shown in Fig. 3A. The traces obtained after averaging at least five individual responses were used to assess the peak amplitude and time constant of decay (Fig. 3B). The decay time constant of NMDACs was generally best described by two exponential curves, with a value for τf of 110 ± 10 ms (range, 38-276 ms) and for τs of 704 ± 49 ms (range, 386-1171 ms); the relative contribution to the peak amplitude of the NMDACs of the fast component was 58 ± 5 %. The value for τw for NMDAC decay was 508 ± 60 ms which was statistically different only from that of eN-EPSCs in the P13-P15 group (P < 0.01, independent t test).

Figure 3. NMDA-mediated currents (NMDACs) in patches from visual cortical neurons in brain slices from P13-P15 rats.

Figure 3

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NMDACs were elicited by a fast application of brief (1 ms) pulses of 1 mM L-glutamate and NBQX (5 μM). The application frequency (0.33 Hz) was adjusted to minimize run-down due to desensitization of NMDA receptors. A, four selected individual examples of NMDACs elicited in the absence (left) and presence (right) of haloperidol. The inset shows an expanded view of a portion of the last record in which single-channel currents can be observed. The drug caused a strong reduction in the open probability and, as shown in the inset, it also strongly decreased open channel current duration. The open tip current used to measure the duration of the drug application pulse is shown above the first current trace in each panel. The amplitude calibration bar does not apply to these open tip currents. B, a mean of five NMDACs in the presence (left) and the absence (right) of haloperidol is shown, with superimposed decay fitting curve together with an indication of the decay time constants. C, open time distributions of channel currents for the patch shown above with superimposed double exponential fitting and an indication of the weighted mean time constants (τw) deriving from the double exponential fitting of these distributions. Because of the high level of channel activity, we were able to assess the channel open time distribution only from selected the regions of each sweep without overlapping channel channel currents.

Haloperidol (50 μM), studied in fourteen neurons, produced a sharp decrease in the peak amplitude of NMDACs (Fig. 3A). The mean reduction observed in the patch population studied was 79 ± 14 %. Haloperidol at this high concentration, as reported on recombinant receptors (Ilyin et al. 1996), also produced a reduction of the mean channel open time, as illustrated in Fig. 3A (inset). Because of the high level of channel activity with glutamate application, it was impossible to perform a conventional analysis of channel current kinetics. However, we were able to assess the channel current amplitude and mean open time distribution by selecting the regions of each sweep without overlapped openings. In Fig. 3C we report an example of the open time distribution of channel currents from one patch before and during haloperidol. The weighted mean time constants (τw) deriving from the double exponential fitting of these distribution in seven patches was decreased by 35 % with 50 μM haloperidol, from 3.5 ± 0.3 to 2.3 ± 0.2 ms. Single-channel current amplitude was not changed (not shown). The observed reduction in open time precluded us from analysing variations in the decay of the mean NMDACs before and after the drug. For comparison with results obtained with N-EPSCs and to further confirm the surprisingly strong blockade of NMDA receptors in thirteen excised outside-out patches, we studied the effects of increasing concentration of CP101,606 on NMDACs. As shown in Fig. 4A, CP101,606 produced a dose-dependent decrease in peak NMDAC amplitude. A summary of the dose-dependent reduction of NMDACs is shown in Fig. 4B. With a concentration of 10 μM CP101,606 we obtained a reduction of 75 ± 5 %, similar to the reduction observed with 50 μM haloperidol, but significantly different (P < 0.01, independent t test) from the reduction in peak eN-EPSC amplitude induced by 10 μM CP101,606 at the same age. As with eN-EPSCs we were unable to obtain full recovery from the effects of CP101,606 at the concentration used due to the length of time required for wash-out of the drug compared with the duration of our patches. However, as illustrated in the example in Fig. 4A, we did observe some recovery on wash-out (67 ± 15 %).

Figure 4. Dose-dependent antagonism by CP101,606 of NMDACs in patches from visual cortical neurons in brain slices from P13-P15 rats.

Figure 4

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A, increasing doses of CP101,606 antagonized NMDACs elicited in an outside-out patch excised from a cortical neuron at P14. τw values derived from the weighted mean of the fast and slow time constants of the double exponential curves that best describe the NMDACs decay are shown for the control response and that observed with 1 μM CP101,606. B, a summary of the dose-dependent reduction produced by increasing doses of CP101,606 in the thirteen patches tested from cortical neurons at P13-P15. The CP101,606 concentration producing half-maximal inhibition was between 1 and 10 μM. C, a summary of the percentage reduction in the parameters characterizing the NMDAC decay produced by 1 μM CP101,606. τf and τs are the fast and slow time constant of the double exponential curves, respectively, that best describe the NMDAC decay. %F is the relative contribution to peak NMDAC of the amplitude of the fast decay component. None of these reductions were significantly different from zero (one-population t test). D, scatter plots of the percentage reduction in the mean amplitude of NMDACs produced by bath perfusion with 10 μM CP101,606 (•) and 50 μM haloperidol (○) as a function of the time constant (τw) derived from the weighted mean of the fast and the slow time constants of a double exponential fitting of the decay of control NMDACs.

Double exponential fitting of the NMDACs before and after perfusion with 1 μM CP101,606 did not reveal significant changes from control values (Fig. 4C). We report, however, the percentage reduction in the peak amplitude of NMDACs produced with NR2B antagonists as a function of the weighted mean (τw) of the two exponential time constants describing the decay of NMDACs. In Fig. 4D it can be observed that antagonism by haloperidol (50 μM) and CP101,606 (10 μM) was not correlated with the initial τw.

DISCUSSION

Differences in decay kinetics of NMDA-mediated excitatory synaptic currents have been proposed to underlie the temporally distinct developmental profile of plastic changes occurring during development in the sensory cortex (Sheetz & Constantin-Paton, 1994; Fox, 1995). N-EPSCs in cortical slices have been previously reported (Carmignoto & Vicini, 1992; Crair & Malenka, 1995; Kim et al. 1995) but correlation with the NMDAR subunit composition and the properties of native receptors has not yet been demonstrated. In our study, we took advantage of specific antagonists of NMDAR subtypes containing the NR1/NR2B subunits, in order to investigate the native NMDAR subunit composition at excitatory synapses in visual cortical neurons at distinct developmental ages.

In situ hybridization data have shown a highly prevalent expression of the NR1/NR2B subunit in the developing cortex during the first 2 weeks after birth in rodents (Monyer et al. 1994; Takahashi et al. 1996). At the same time, recombinant NMDA receptors comprising NR1/NR2B subunits have been characterized by slow deactivation kinetics (Monyer et al. 1994; Li et al. 1996). Therefore, we were expecting mN-EPSCs characterized by slow decay kinetics and sensitivity to NR1/NR2B-specific blockers in rats at this age.

mN-EPSCs in developing visual cortical neurons and NR1/NR2B-specific blockers

Spontaneous mN-EPSCs occurred at a low frequency in most neurons investigated. They were of small amplitude and hence very easily confused with the background noise. In spite of these limitations, clear synaptic currents were often observed and, although the number of samples was limited, we could study the amplitude and frequency of occurrence of mN-EPSCs. We compared mN-EPSCs in neurons from the visual cortex at P7-P8 and P13-P15. In the majority of neurons from rats at P7-P8, mN-EPSCs were not detectable and in those neurons where mN-EPSCs were recorded, they were characterized by a significantly smaller amplitude than currents in neurons at P13-P15. This observation is consistent with a developmental increase of release sites and of receptor density at these sites. In contrast to our expectations following reports of high expression of the NR2B subunit in the developing cortex, haloperidol and CP101,606, specific blockers of receptors comprising NR1/NR2B subunits (Chenard et al. 1995; Ilyin et al. 1996), failed to antagonize mN-EPSCs in the majority of cells in the visual cortex at P13-P15. While CP101,606 never produced significant changes in the mean mN-EPSC amplitude, haloperidol produced significant alterations in a few of the neurons tested. The increase in the mean mN-EPSC amplitude observed in some cells with haloperidol may, however, relate to dopamine receptor antagonism since it was also observed in several cells with the selective D1 antagonist spiperone.

An observation which was even more unexpected was the lack of effect of CP101,606 on mN-EPSCs in visual cortical slices taken from animals as young as P7-P8, the age at which in situ hybridization shows a clear prevalence in the expression of mRNA for the 2B subunit in the cortex (Monyer et al. 1994; Mori & Mishina, 1995). Our data suggest that neither at P13-P15 nor at P7-P8 were ‘pure’ NR1/NR2B receptors important in excitatory synapses. A possible interpretation of these findings is that the NR2B subunits were associated with other subunits (e.g. NR2A or NR2D), and therefore haloperidol and CP101,606 antagonism of the receptors was reduced. This hypothesis is supported by evidence obtained from co-transfection of mammalian kidney cells with mixtures of NR1, NR2A and NR2B subunit cDNAs. Under these conditions the formation of recombinant receptors characterized by slow deactivation kinetics and with no sensitivity to haloperidol was observed (Li et al. 1996). Based on this observation, we speculate that even a limited presence of NR2A subunits (as is found at early developmental stages) is capable of preventing NR2B-selective antagonism. In cells at P7-P8, CP101,606 failed to antagonize mN-EPSCs but it produced a significant reduction in the frequency of their occurrence. This result may relate to the existence of a larger number of synaptic sites comprising ‘pure’ NR1/NR2B receptors at P7-P8 than at P13-P15.

Pharmacological antagonism of evoked N-EPSCs

We also investigated the effects of pharmacological antagonists on evoked N-EPSCs in developing visual cortical neurons for comparison with the effects observed on mN-EPSCs. This approach allowed us to extend our investigation of synaptic NMDA receptors to rats at a very early postnatal age (P3-P5) in which no spontaneous synaptic events were detectable. For this investigation we used CP101,606, since both haloperidol and ifenprodil were shown to produce blockade of voltage-gated Ca2+ conductances (Fletcher et al. 1994; Church et al. 1994) and thereby elicit presynaptic effects. The possibility that the maximal CP101,606 concentration used in our experiments produced presynaptic actions was ruled out by studying its effects on AMPA receptor-mediated EPSCs.

As we observed for mN-EPSCs in neurons at P7-P8 and at P13-P15, CP101,606 failed to depress the mean amplitude of eN-EPSCs in both age groups. In the neuron group at P3-P5, CP101,606 significantly reduced the mean amplitude of eN-EPSCs and it was effective in most neurons tested. Taken together, our results indicate that at P3-P5 a consistent proportion of synapses contains ‘pure’ NR1/NR2B heteromers and with development the receptor subunit composition at these synapses changes to one that does not exhibit sensitivity to NR2B blockers. Since the mRNA for NR2D subunits decreases with development, it is unlikely that the occlusion of the antagonistic effects of NR1/NR2B blockers at P7-P8 and P13-P15 is due to the formation of NR1/NR2B/NR2D subunit assemblies in the postsynaptic receptors. This further supports our proposal that the NR2A subunit is a critical element in the development of excitatory synapses as early as P7.

The degree of antagonism of eN-EPSCs at P3-P5 is smaller than that observed in excised patches at P13-P15. Although we have no reason to believe that penetration of the slices by CP101,606 was poor, we cannot completely rule out the possibility that a decrease in the concentration of this blocker may account for the decrease in eN-EPSC antagonism at P3-P5. However, it is difficult to attribute the lack of antagonism of eN-EPSCs observed in neurons at P13-P15 solely to this effect. Furthermore, it is still possible that at P3-P5 NR1/NR2D or NR1/NR2B/NR2D heteromers make up a proportion of the synaptic receptors.

While overall CP101,606 failed to depress the mean amplitude of mN-EPSCs and eN-EPSCs at P7-P8 and P13-P15, in a few cells at P13-P15 and in a more consistent number of cells at P7-P8, CP101,606 produced a significant reduction in eN-EPSC amplitude but not of mN-EPSC amplitude. The difference we observed in our comparison between distinct synaptic events (mN-EPSCs vs. eN-EPSCs) may relate to the fact that these events are generated at distinct synapses by the activation of different NMDA receptors. This hypothesis is also supported by the decrease in the frequency of mN-EPSCs observed in cells at P7-P8. In this age group most cells do not exhibit mN-EPSCs, although they all respond to electrical stimulation, leading to a selection of the cell population in the mN-EPSC study.

Pharmacological antagonism and decay kinetics of evoked N-EPSCs

We investigated the decay of eN-EPSCs in all age groups. As reported in other brain regions, this decay is best described by a double exponential curve (Carmignoto & Vicini, 1992; Spruston, Jonas & Sakmann, 1995; Plant et al. 1997). To allow a rapid comparison among various experimental conditions we used a time constant (τw) derived from the weighted mean of the fast and the slow time constants. τw was significantly faster in the cortical neuron group studied at P13-P15 than at younger ages, consistent with a developmental reduction in NR2D subunits and an increase in NR2A subunits (Flint et al. 1997). Furthermore, we observed some correlation between τw and the extent of blockade by CP101,606 when cells studied at all age groups were pooled together. However, CP101,606 failed to alter the τw characterizing eN-EPSC kinetics at all ages tested and no correlation between τw and CP101,606 blockade was observed within each age group. These results were surprising because we expected from previous studies which relate the presence of NR1/NR2B to slow decay kinetics (Monyer et al. 1994; Li et al. 1996) that we should have observed a stronger NR2B antagonist block in cells with a slower τw at all ages considered. Furthermore, if a consistent population component of ‘pure’ NR1/NR2B receptors coexisted with NR1/2A receptors at synaptic sites we should have observed a shortening of eN-EPSC decay with CP101,606. To explain our observations, the possibility should be considered that a large proportion of receptors contains other heteromers (such as those containing both NR2A and NR2B subunits), and that these heteromers are characterized by a slow deactivation and insensitivity to NR2B blockers (Li et al. 1996; Boeckman & Aizenman, 1996). Indeed, Luo, Wang, Yasuda, Dunah & Wolfe (1997) have recently reported that, in the adult rat cerebral cortex, the amount of assembled NR1/NR2A binary complex is quite small relative to NR1/NR2B and NR1/NR2A/NR2B complexes. We should also consider, however, that the lack of correlation between deactivation kinetics and pharmacological sensitivity in distinct age groups might not be related only to distinct deactivation properties of NMDA receptors with different subunit compositions; rather it may reflect possible post-translational modifications that can produce longer series of channel openings (Lieberman & Mody, 1994; Yu, Askalan, Keil & Salter, 1997).

Recently, antagonism of hippocampal NMDA-mediated synaptic currents by ifenprodil has been shown to be dependent on development (Kirson & Yaari, 1996), implying a developmental decrease in synaptic receptors comprising the NR2B subunit. At the same time, two reports have attempted to correlate the presence of mRNA for NMDA receptor subunits with the decay kinetics of eN-EPSCs in neurons in brain slices (Flint et al. 1997; Plant et al. 1997). The results of these studies demonstrate that the abundant presence of the NR2A subunit shortens eN-EPSC duration in developing hippocampal neurons (Flint et al. 1997) but it is not sufficient to produce fast deactivation kinetics in medial septal neurons (Plant et al. 1997). Interestingly, in septal neurons, while the variable sensitivity to blockade by ifenprodil indicates the presence of heteromers of NR2A and NR2B receptor subunits, the decay kinetics of eN-EPSCs is always dominated by the NR2B subunit.

NMDA receptor currents in excised outside-out patches

Currents produced by the rapid activation of somatic NMDA receptors in visual cortical neurons at P13-P15 in excised membrane patches (NMDACs) displayed double exponential decay as previously shown in several brain regions (Carmignoto & Vicini, 1992; Spruston, Jonas & Sakmann, 1995; Gotz, Kraushaar, Geiger, Lubke, Berger & Jonas, 1997). To allow an easier comparison between the decay of NMDACs and eN-EPSCs at different age groups and with different drug treatments we used τw. We observed a significant difference between τw for NMDACs and eN-EPSCs at P13-P15. On the other hand, in patches at P13-P15 NMDA receptor kinetics was similar to that of synaptic receptors at P7-P8 and P3-P5. We also observed a strong blockade with both haloperidol and CP101,606 of NMDACs in all patches tested in visual cortical neurons at P13-P15. Our data allow us to speculate that the subunit composition of somatic NMDA receptors is mostly NR1/NR2B with little or no contribution to the receptor assembly of the NR2A subunit at P13-P15. Since we studied NMDA receptors in outside-out patches excised from the soma of large visual cortical neurons, where excitatory synapses are rarely found (Peters, 1985), our results support the hypothesis of differences between synaptic and extrasynaptic receptors (Clark, Farrant & Cull-Candy, 1997). Spruston et al. (1995) reported that the functional properties of native receptors in dendrites of hippocampal neurons were dominated by the presence of the NR2B subunit. However, they failed to observe differences in kinetic properties between somatic and dendritic patches which, given the high density of spines in hippocampal CA1 and CA3 pyramidal neurons, should probably have contained at least some synaptic receptors. The latter results could be explained if a receptor population was characterized by slow deactivation kinetics and lower NR2B blocker sensitivity such as we observed at synapses in neurons at P7-P8.

Acknowledgments

This work supported by NINDS grants P01 NS28130 and K04 NS01680 to S. V.

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