Synapse-specific expression of functional presynaptic NMDA receptors in rat somatosensory cortex

Abstract

Presynaptic NMDA receptors (NMDARs) modulate release and plasticity at many glutamatergic synapses, but the specificity of their expression across synapse classes has not been examined. We found that non-postsynaptic, likely presynaptic NR2B-containing NMDARs enhanced AMPA receptor-mediated synaptic transmission at layer 4 (L4) to L2/3 (L4-L2/3) synapses in juvenile rat barrel cortex. This modulation was apparent at room temperature when presynaptic NMDARs were activated by elevation of extracellular glutamate or application of exogenous NMDAR agonists. At near physiological temperatures, modulation of transmission by presynaptic NMDARs occurred naturally, without the need for external activation. Blockade of presynaptic NMDARs depressed unitary and extracellularly evoked EPSCs at L4-L2/3 synapses, accompanied by increases in paired-pulse ratio and coefficient of variation, indicative of a decrease in presynaptic release probability. NMDAR agonists increased the frequency of miniature EPSCs in L2/3 neurons, without altering their amplitude or kinetics. Focal application of NMDAR antagonist revealed that the NMDARs that modulate L4-L2/3 transmission are located in L2/3, not L4, consistent with localization on terminals or axons of L4-L2/3 synapses, rather than on the somatodendritic compartment of presynaptic L4 neurons. In contrast, presynaptic NMDARs did not modulate L4-L4 synapses, which originate from the same presynaptic neurons as L4-L2/3 synapses, or cross-columnar L2/3-L2/3 horizontal projections, which synapse onto the same postsynaptic target neurons. Thus, presynaptic NMDARs selectively modulate L4-L2/3 synapses, relative to other synapses made by the same neurons. Existence of these receptors may support specialized processing or plasticity by L4-L2/3 synapses.

Figure 1.

Non-postsynaptic NMDARs modulate synaptic transmission at L4–L2/3 synapses in S1. A, Representative recording showing that bath application of d-APV reduces the amplitude of AMPA-EPSCs at presumptive L4–L2/3 synapses activated by extracellular L4 stimulation. This recording was made in 15 μm TBOA, focal BMI, and internal MK-801 to block postsynaptic NMDA receptors. Top, Amplitude of the first AMPA-EPSC throughout the recording for this cell. Inset, Pair of AMPA-EPSCs (30 Hz) during baseline (black), d-APV application (gray) and after d-APV washout (dashed black). Each trace in this and other figures is the mean of the last 10 sweeps in each condition. Bottom, Holding current, series resistance, and input resistance. B, Mean effect of d-APV on amplitude of first AMPA-EPSC for five cells. Error bars indicate SEM. d-APV (50 μm) was applied for 14 min. C, Mean effect of 30 min d-APV application on first AMPA-EPSC amplitude (50 μm d-APV, n = 5 cells, filled squares; control experiments, n = 5 cells, open squares). D, Effect of d-APV on PPR for 14 min (filled diamonds) and 30 min (filled squares) applications. Open symbols, PPR for corresponding time points during control experiments in which d-APV was not applied. **p < 0.01; *p < 0.05.

Figure 2.

Activation of PreNMDARs increases mEPSC frequency in L2/3 pyramidal neurons. A, Representative mEPSCs from a L2/3 pyramidal neuron during baseline (top), 15 μm NMDA application (middle), and 15 μm NMDA plus 50 μm d-APV application (bottom). Recordings were made in 500 nm TTX, 100 μm picrotoxin, and iMK-801. B, Cumulative probability histogram for inter-mEPSC interval during baseline (solid black), NMDA (gray), and NMDA plus APV (dashed black) conditions. The histogram is a mean of individual histograms from six cells (p < 0.01 for baseline vs NMDA and NMDA vs NMDA plus APV; p > 0.05 for baseline vs NMDA plus APV, Kolmogorov–Smirnov test). Inset, Average mEPSC waveform across the population (black, baseline; gray, NMDA; dashed, NMDA plus APV). C, Mean mEPSC frequency for each cell. Error bars represent the population mean. **p < 0.01. D, Mean mEPSC amplitude for each cell.

Figure 3.

Focal application of d-APV reveals that PreNMDARs at the L4–L2/3 projection are located in L2/3, not in L4. A–F, AMPA-EPSCs were measured in L2/3 pyramidal cells in response to stimulation in L4, while d-APV was focally applied via a puffer pipette in either L2/3 (A–C), or in L4 (D–F). A, Experiment schematic for experiments in which d-APV was focally applied via a puffer pipette in L2/3. Inset, Postsynaptic NMDA-EPSCs measured from a representative L2/3 neuron (top trace pair) and L4 neuron (bottom trace pair) before (black) and during (gray) puffer application of 2.5 mm d-APV in L2/3. Each trace shown is the average of the last 10 sweeps of each condition. Focal d-APV application blocked NMDA currents strongly in L2/3, but only modestly in L4. B, Example cell showing the effect of focal puffing of d-APV in L2/3 on AMPA-EPSCs on the L4–L2/3 projection. Top, Amplitude of the first of a pair of AMPA-EPSCs. Inset, Pairs of AMPA-EPSCs during baseline (black), d-APV puffing (gray), and after recovery (black, dashed). Each trace shown is the average of the last 10 sweeps of each condition. Bottom, Holding current, series resistance, and input resistance for this recording. C, Mean effect of d-APV puffing in L2/3 on AMPA-EPSCs (n = 12 cells, filled circles), and effect of puffing Ringer's solution in L2/3 instead of d-APV (n = 5, open circles). D–F, Equivalent experiments on nine cells in which d-APV was puffed in L4, rather than in L2/3. G, Effect on PPR of focal puffing of DAPV in L2/3 (filled circles), Ringer's in L2/3 (open circles), and d-APV in L4 (open squares). Error bars represent population means. *p < 0.05.

Figure 4.

PreNMDARs preferentially contain NR2B subunits. A, Blockade of pharmacologically isolated postsynaptic NMDA-mediated EPSCs by increasing ifenprodil concentration. Inset, Representative traces from a single cell during baseline (solid black), 3 μm (solid gray), 4 μm (dashed black), and 6 μm ifenprodil (dashed gray). B, Effect of 3 μm ifenprodil on amplitude of the first EPSC for a representative cell. Inset, Pairs of EPSCs before (black) and during (gray) 3 μm ifenprodil. Each trace shown is the average of the last 10 sweeps of each condition. C, Group data for cells with 3 μm ifenprodil application (n = 10, filled circles) and for control experiments without ifenprodil (n = 8; open circles). D, Change in PPR for 3 μm ifenprodil application (filled circles) and control (open circles). Error bars represent population means. **p < 0.01. E, Representative mEPSCs from a single L2/3 pyramidal neuron during baseline, 20 μm HQA application, and 20 μm HQA plus 3 μm ifenprodil. F, Mean cumulative probability histogram for inter-mEPSC interval during baseline (solid black), HQA (gray), and HQA plus ifenprodil (dashed black; p < 0.01 for baseline vs HQA and for HQA vs HQA plus ifenprodil; p > 0.05 for baseline vs HQA plus ifenprodil, Kolmogorov–Smirnov test). Inset, Mean frequency for each cell during each condition. Error bars represent population mean. *p < 0.05; **p < 0.01.

Figure 5.

PreNMDARs are located on the L4–L2/3 projection but not on other cortical inputs to L2/3 pyramidal cells. A, Representative experiment testing the effect of d-APV on AMPA-EPSCs evoked on the L2/3 cross-columnar pathway (L2/3–L2/3) and on L4–L2/3 inputs to the same postsynaptic cell. Responses to the two pathways were measured in alternation. Top plots, Amplitude of the first AMPA-EPSC on each pathway. Insets, Pairs of AMPA-EPSCs before (black) and during (gray) d-APV application. Each trace shown is the average of the last 10 sweeps of each condition. Bottom plots, Holding current, series resistance, and input resistance for this cell. B, Recording set-up for these experiments. C, Mean effect of d-APV on first AMPA-EPSC amplitude for L2/3–L2/3 inputs (triangles) and simultaneously measured L4–L2/3 inputs (circles) (n = 8 cells). D, PPR changes in this experiment. Error bars represent population means. *p < 0.05.

Figure 6.

Functional PreNMDARs are absent from local synapses between L4 excitatory cells. A, Differential interference contrast image of example synaptically coupled L4 excitatory cells, with regular-spiking pattern for these cells. B1, Postsynaptic uEPSCs elicited by a pair of presynaptic spikes before (black) and after (gray) 50 μm d-APV application for the regular-spiking pair above. Each trace shown is the average of the last 10 sweeps of each condition. B2, Top, Lack of effect of d-APV on amplitude of the first uEPSC for one representative cell pair. Bottom: Postsynaptic holding current, postsynaptic series resistance, postsynaptic input resistance, presynaptic membrane potential, and presynaptic input resistance for this pair. C, Mean effect of d-APV application on first uEPSC amplitude (n = 7 pairs). D, Effect of d-APV on PPR. Error bars show population means. E, Mean mEPSC frequency for five L4 cells before and after 20 μm HQA application. F, Cumulative probability histogram of mEPSC interval before (black) and after (gray) HQA application (p > 0.1, Kolmogorov–Smirnov test). Inset, Mean mEPSC before (black) and during (gray) HQA application.

Figure 7.

PreNMDARs regulate transmission at synaptically coupled L4–L2/3 pairs. A1, Example AMPA-uEPSCs from one coupled presynaptic L4 cell and postsynaptic L2/3 cell in response to a pair of presynaptic spikes during baseline (solid black) and 50 μm d-APV (gray). Each trace shown is the average of the last 10 sweeps of each condition. Inset, Regular-spiking pattern for presynaptic L4 cell. A2, Top, Effect of d-APV on first AMPA-uEPSC amplitude for this cell pair. Bottom, Postsynaptic holding current, postsynaptic series resistance, postsynaptic input resistance, presynaptic membrane potential, and presynaptic input resistance for this pair. B, Mean effect of d-APV on first AMPA-uEPSC amplitude across seven cell pairs (filled circles) and across four control cell pairs without d-APV (open circles). C, Change in PPR caused by d-APV application (filled circles) and for control pairs (open circles). Error bars represent population means. *p < 0.05. D, CV analysis of effects of d-APV on first AMPA-uEPSC amplitude. Small filled circles, CV⁻² versus mean EPSC amplitude for each pair in which 50 μm d-APV was applied. Large filled circle, Population average and SE for these pairs. Large open circle, Average effect on control pairs with no d-APV application. All values are normalized to the baseline period for each pair.

Figure 8.

PreNMDARs are active, but not saturated, at 30–32°C in the absence of TBOA. A1, Single example of depression of extracellularly evoked L4–L2/3 synaptic responses by 50 μm d-APV at 30–32°C, without TBOA or exogenous NMDAR agonist. Focal BMI and iMK-801 were included to isolate AMPA-EPSCs. The first EPSC amplitude is shown throughout the recording. Inset, Pairs of AMPA-EPSCs (30 Hz) during baseline (black), d-APV application (gray), and after d-APV washout (dashed black). Each trace is mean of the last 10 sweeps in each condition. A2, Mean effect of d-APV on amplitude of first AMPA-EPSC at L4–L2/3 synapses (n = 7; filled symbols) and at extracellularly evoked L2/3–L2/3 cross-columnar synapses (n = 7; open symbols). Bars are SEM. A3, Effects of d-APV on PPR for cells in (A2). B1, d-APV (50 μm) decreased mEPSC frequency in L2/3 pyramidal cells at 30–32°C. B2, Cumulative probability histogram for inter-mEPSC interval during baseline (solid black) and d-APV (gray; p < 0.01, Kolmogorov–Smirnov test). Inset, Mean mEPSC before (black) and during (gray) d-APV application. C1, Effects of HQA (20 μm) and subsequent APV (50 μm) on mEPSC frequency in L2/3 pyramidal cells. C2, Cumulative probability histogram for inter-mEPSC interval during baseline (solid black), HQA (gray), and HQA plus APV (dashed black); (p < 0.01, Kolmogorov–Smirnov test for all pairwise comparisons). Inset, Mean mEPSC during baseline (black), HQA (gray), and HQA plus APV (dashed black). D1, HQA (20 μm) and HQA plus d-APV (50 μm) do not affect mEPSC frequency in L4. D2, Cumulative probability histogram for inter-mEPSC interval during baseline (solid black), HQA (gray), and HQA plus APV (dashed black); (p > 0.05, Kolmogorov–Smirnov test for all pairwise comparisons). Inset, Mean mEPSC during baseline (black), HQA (gray), and HQA plus APV (dashed black). *p < 0.05; **p < 0.01.