GCP II (NAALADase) inhibition suppresses mossy fiber-CA3 synaptic neurotransmission by a presynaptic mechanism

Abstract

We tested the hypothesis that endogenous N-acetylaspartylglutamate (NAAG) presynaptically inhibits glutamate release at mossy fiber-CA3 synapses. For this purpose, we made use of 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), an inhibitor of glutamate carboxypeptidase II [GCP II; also known as N-acetylated alpha-linked acidic dipeptidase (NAALADase)], the enzyme that hydrolyzes NAAG into N-acetylaspartate and glutamate. Application of 2-MPPA (1-20 microM) had no effect on intrinsic membrane properties of CA3 pyramidal neurons recorded in vitro in whole cell current- or voltage-clamp mode. Bath application of 10 microM 2-MPPA suppressed evoked excitatory postsynaptic current (EPSC) amplitudes. Attenuation of EPSC amplitudes was accompanied by a significant increase in paired-pulse facilitation (50-ms interpulse intervals), suggesting that a presynaptic mechanism is involved. The group II metabotropic glutamate receptor (mGluR) antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l) propanoic acid (LY341495) prevented the 2-MPPA-dependent suppression of EPSC amplitudes. 2-MPPA reduced the frequencies of TTX-insensitive miniature EPSCs (mEPSC), without affecting their amplitudes, further supporting a presynaptic action for GCP II inhibition. 2-MPPA-induced reduction of mEPSC frequencies was prevented by LY341495, reinforcing the role of presynaptic group II mGluR. Because GCP II inhibition is thought to increase NAAG levels, these results suggest that NAAG suppresses synaptic transmission at mossy fiber-CA3 synapses through presynaptic activation of group II mGluRs.

FIG. 1

The glutamate carboxypeptidase II [GCPII, N-acetylated alpha-linked acidic dipeptidase (NAALADase)] inhibitor 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, 10 μM) does not affect intrinsic membrane properties of CA3 pyramidal neurons. A: voltage recordings of responses to current pulses (400-ms duration; −120–140 pA) before (control) and 10 min after drug application (2-MPPA). B: whole cell currents recorded in voltage-clamp mode. Numbered triangles depict time points analyzed in the I-V plots below. 2-MPPA has no effect on transient outward currents (B1), inwardly rectifying currents (B2 and B3), or sustained currents (B3).

FIG. 2

2-MPPA suppresses mossy fiber-CA3 evoked excitatory postsynaptic currents (EPSCs) and enhances paired-pulse facilitation. A1: time course of amplitudes of the first of a pair of EPSC recorded in response to a pair of stimuli (20 Hz) before, during, and after application of 2-MPPA (10 μM). Representative traces are depicted above. Bottom graph plots the time course of the normalized paired-pulse ratios. A2: 2-MPPA does not affect input resistance. Superimposed responses recorded before (—) and after (- - -) drug application, in response to 2-mV hyperpolarizing pulses applied before stimuli (◂, stimulus artifact). A3: superimposed traces, scaled to the amplitude of the 1st control EPSC, demonstrate the 2-MPPA-evoked enhancement of paired-pulse facilitation. Roman numerals correspond to time points indicated on amplitude graph. A4: dose-response curve for EPSC depression (% control) produced by 2-MPPA, showing saturation at concentrations <10 μM, IC₅₀ = 1.33. Values are means ± SE (n = 4– 8 cells). B: group data (n = 8 cells) of normalized EPSC amplitudes and paired-pulse ratios (average of 5 consecutive traces). C: mean ± SE changes in EPSC amplitudes and paired-pulse rations (PPR). *, P < 0.001 (ANOVA).

FIG. 3

EPSCs recorded in the presence of [Ca²⁺]/[Mg²⁺] ratio = 3:1. Under this condition, paired-pulse facilitation (PPF) was converted to paired-pulse depression (PPD, A1). 2-MPPA reversed the PPD to PPF (A2). B: Summary data of effect of 2-MPPA on paired pulse ratio in 5 experiments.

FIG. 4

GCP II inhibition presynaptically suppresses transmitter release by acting on type II mGluRs. A1: representative EPSCs demonstrate that preincubation with the mGluR antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l) propanoic acid (LY341495, 100 nM) reversibly suppressed the effects of 2-MPPA. Roman numerals refer to time points in A2. Time courses of EPSC amplitudes and paired-pulse ratio (PPR) are depicted in A2, and superimposed responses, scaled to the 1st EPSC, are shown in A3. B: time course of group data for normalized mean ± SE EPSC amplitudes and PPR (n = 10 cells). Mean ± SE (10 cells) normalized EPSC amplitudes (B) and PPR (C; ANOVA, *: P < 0.001).

FIG. 5

GCP II inhibition reduces miniature EPSC (mEPSC) frequency without affecting their amplitudes. A: mEPSC recorded during control artificial cerebrospinal fluid (ACSF; top) and 10 min after 2-MPPA application (10 μM; bottom). Representative EPSCs are shown at higher scale. The amplitude distribution of these mEPSCs is shown in A1 (n = 148 events for control and 91 events for 2-MPPA). Superimposed and scaled average traces (A2) and cumulative probability plots (A3; K-S test, P > 0.05) demonstrate that 2-MPPA had no effect on mEPSC kinetics. By contrast, 2-MPPA significantly suppressed the frequency of mEPSCs (A and A4; K-S test, P < 0.01). Group data (n = 10 cells; B–D) support this conclusion.

FIG. 6

The effects of GCP II inhibition on mEPSCs are mediated by presynaptic, type II mGluRs. Representative records of mEPSCs (A) and group data (B–D; n = 6 neurons) demonstrate that the mGluR antagonist LY341495 (100 nM) reversibly suppressed the effects of 2-MPPA on the frequency of mEPSCs. B and C: cumulative probability plots of mESPC amplitudes and frequencies. D: the mean ± SE of mEPSC amplitudes and frequency (ANOVA, *: P < 0.01).