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. Author manuscript; available in PMC: 2010 Mar 11.
Published in final edited form as: Synapse. 2009 Apr;63(4):308–318. doi: 10.1002/syn.20609

Cholinergic direct inhibition of N-methyl-D aspartate receptor-mediated currents in the rat neocortex

Jorge Flores-Hernandez #,@, Humberto Salgado $,@, Victor De la Rosa #, Tania Avila-Ruiz #, Oswaldo Torres-Ramirez #, Gustavo Lopez-Lopez #, Marco Atzori $,&
PMCID: PMC2836798  NIHMSID: NIHMS133067  PMID: 19140165

Abstract

Acetylcholine (ACh) and N-methyl-D aspartate receptors (NMDARs) interact in the regulation of multiple important brain functions. NMDAR activation is indirectly modulated by ACh through the activation of muscarinic or nicotinic receptors. Scant information is available on whether ACh directly interacts with the NMDAR. By using a cortical brain slice preparation we found that the application of acetylcholine as well of other drugs acting on muscarinic or nicotinic receptors induces an acute and reversible reduction of NMDAR-mediated currents (INMDA), ranging from 20% to 90% of the control amplitude. The reduction displayed similar features in synaptic INMDA in brain slices, as well as in currents evoked by NMDA application in brain slices or from acutely dissociated cortical cells, demonstrating its postsynaptic nature. The cholinergic inhibition of INMDA displayed an onset-offset rate in the order of a second, was resistant to the presence of the muscarinic antagonist atropine (10 μM) in the extracellular solution, and of G-protein blocker GDPβS (500 μM) and activator GTPγS (400 μM) in the intracellular solution, indicating that it was not G-protein dependent. Recording at depolarized or hyperpolarized holding voltages reduced NMDAR-mediated currents to similar extents, suggesting that the inhibition was voltage-independent, while the reduction was markedly more pronounced in the presence of glycine (20 μM). A detailed analysis of the effects of tubocurarine suggested that at least this drug interfered with glycine-dependent NMDAR-activity.

We conclude that NMDAR-mediated currents can be inhibited directly by cholinergic drugs, possibly by direct interaction within one or more subunits of the NMDAR. Our results could supply a new interpretation to previous studies on the role of ACh at the glutamatergic synapse.

Keywords: acetylcholine, NMDA, patch-clamp, auditory cortex, muscarinic, nicotine

Introduction

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory transmission and are involved in synaptic function associated with long-term synaptic changes, oscillations, bursting behavior, learning, and memory (Burgos-Robles et al., 2007; Castellano et al., 2001; Chub et al., 1998; Nicoll and Malenka, 1999). Acetylcholine (ACh) released from terminals of neurons whose cell body are located in the Nucleus Basalis (Voytko, 1996; Voytko et al., 1994) modulates the behavior of many cortical areas by activation of muscarinic receptors (Mash and Potter, 1986) and nicotinic receptors (Alkondon and Albuquerque, 2004). Excitatory synaptic transmission in the primary auditory neocortex is subject to a particularly intense cholinergic modulation (Cox et al., 1992; Metherate and Ashe, 1991; Metherate and Ashe, 1993a; Metherate and Ashe, 1993b; Metherate and Ashe, 1994; Metherate et al., 1990; Metherate and Weinberger, 1989; Metherate and Weinberger, 1990). Cholinomimetics appear to have multiple effects on NMDAR-mediated currents (INMDA): ACh increases INMDA by acting on muscarinic and nicotinic receptors located in presynaptic terminals during the early postnatal period (P8-16) (Aramakis et al., 1997; Aramakis et al., 1999; Aramakis and Metherate, 1998), but decreases INMDA in the retina (O'Dell and Christensen, 1988) and in primary neocortical neuronal cultures (Aizenman et al., 1991).

Less information is available on the cholinergic regulation of INMDA after early development. In particular, it is not clear whether cholinomimetics interact directly with the NMDAR channel or rather the cholinergic inhibition is mediated by second messengers. By studying the cholinergic modulation of INMDA in juvenile rats (P23-45) we found that ACh and other cholinergic, muscarinic and nicotinic agonists and antagonists acutely and reversibly inhibited INMDA. The mechanism of the inhibition appeared to be postsynaptic, resulting directly from the interaction of the cholinergic drug or neurotransmitter with the NMDAR, and was not blocked by a G-protein blocker nor by the presence of the intracellular Ca2+-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid K (BAPTA), suggestive of a direct interaction between cholinergic drugs and the NMDAR.

Methods

Preparation

We used an auditory cortex slice preparation similar to ones previously described (Atzori et al., 2005; Atzori et al., 2001). In some recordings we used a medial prefrontal cortex slice preparation for a series of control experiments, as described previously (Atzori et al., 2005; Atzori et al., 2001). Twenty three-forty five day-old Sprague Dawley rats (Charles River, Wilmington, MA) were anesthetized with forane (Baxter, Round Lake, IL), sacrificed according to the National Institutes of Health guidelines, and their brains sliced with a vibratome in a refrigerated solution (0°-4°C) containing (mM): 130 NaCl, 3.5 KCl, 10 Glucose, 25 NaHCO3, 1.25 NaH2PO4, 1.5 CaCl2 and 1.5 MgCl2, saturated with a mixture of 95% O2 and 5% CO2 (artificial cerebro-spinal fluid, ACSF). The recording solution contained also picrotoxin (100 μM) or bicuculline (10 μM) for blocking γ-aminobutyric acid A receptor (GABAAR), dinitroquinoxaline (DNQX, 10 μM) and atropine (10 μM), for blocking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) -mediated currents, and muscarinic receptors, respectively, throughout the whole study. Coronal slices from the most caudal fourth of the brain were retained after removing the occipital convexity, and subsequently incubated in ACSF at 32°C before being placed in the recording chamber. The recording area was selected dorsally to the rhinal fissure corresponding to the auditory cortex (Rutkowski et al., 2003).

Neuron dissociation

Brain slices were placed in an oxygenated cell-stir chamber containing papain (0.5 mg/ml, Sigma) at 35°C bubbled with O2, titrated at pH = 7.4 with NaOH, 300-305 mOsm/l. After 20 min of enzyme digestion, tissue was rinsed three times with the low-Ca2+-isethionate solution and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish mounted on the stage of an upright fixed-stage microscope containing (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) -buffered ACSF.

Drugs and solutions

All drugs were purchased from Sigma (St. Louis, MO) or from TOCRIS (Ellisville, MO). In order to assess a possible postsynaptic modulation, in some experiments, pulses of N-methyl-D aspartate (NMDA, 100 μM), were applied at 100-200 μM from the recording areas, once every 30 s. NMDA was dissolved in distilled H2O and diluted 10-fold in ACSF before being back-filled to a glass pipette similar to the one used for recording. NMDA application was performed with a pressure system (picospritzer, General Valve Corp. Fairfield, NJ) through a glass pipette (≅ 25 psi, 3-12 ms). Stock solutions of all drugs were prepared in water and drugs were either bath-applied or dispensed with a solenoid valve system with manifold application system with a dead-time of < 200 ms as measured in dissociated cells.

Electrophysiology

Slice recording

Slices were placed in an immersion chamber, where cells with a prominent apical dendrite, suggestive of pyramidal morphology, were visually selected using an Olympus BX51 (Olympus, Tokyo, Japan) with Nomarski optics and an infrared camera system (DAGE-MTI, Michigan City, IN). Excitatory postsynaptic currents (EPSCs) were recorded in the whole-cell configuration, in voltage clamp mode, with 3-5 MΩ electrodes filled with a solution containing (mM): 90 CsCl, 90 gluconic acid, 1 lidocaine N-ethyl bromide (QX314), 1 MgCl2, 10 HEPES, 4 glutathione, 1.5 ATPMg2, 0.3 GTPNa2, 8 biocytin, 5 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid K (BAPTA-K), pH 7.2-7.3 with NaOH and 275 mOsm/l. BAPTA blocks second messenger cascades caused by raises in intracellular calcium concentration (Beech, 1993; Jones et al., 1988; Vulfius et al., 1998). The holding voltage was not corrected for the junction potential (< 4 mV). Electrically evoked EPSC were measured by delivering two electric stimuli (90-180 μs, 10-50 μA) 100 ms-apart every 20 seconds with an isolation unit, through a glass stimulation monopolar electrode filled with artificial cerebrospinal fluid (ACSF) and placed in layer II/III, 150-200 μm from the recording electrode. EPSCs were blocked by aminophosphonovaleric acid (APV, 100 μM), confirming that they were mediated by NMDARs. Paired Pulse Ratio (PPR) was defined as the ratio between the mean of the second to the mean of the first response. INMDA were induced by applications of NMDA itself (chemically-evoked EPSCs (cEPSCs) in slices, in the presence of the Na+-channel blocker tetrodotoxin (TTX, 1 μM), or from dissociated cell.

Recordings started after a stabilization period of a few minutes during which the pipette solution dialyzed into the recorded cell. We defined as statistically stable period a time interval along which the EPSC mean amplitude measured during any two-minute assessment did not vary according to an unpaired t-Student test. After recording an initial baseline for more than 5 minutes, drugs were bath-applied for 5 minutes or longer, until reaching a stable condition (as defined above). In the case of recordings with a pipette solution containing the G-protein blocker GDPβS or activator GTPγS we waited at least 20 min (on average ~ 30 min) before starting electrophysiological recording, in order to allow sufficient time to dialyze the cytosol with the intracellular solution. Drug effects were assessed by measuring and comparing changes between baseline (control) and treatment, with paired t-Student tests (PPR, Wilcoxon's t-test). Analysis of variance (ANOVA) or unpaired t-Student test were used for comparisons between different groups of cells. Data were reported as different only if P < 0.05. We indicated as Acontr and Atreat(ment) the mean amplitude of the glutamatergic currents in control or under the described treatment. The reduction was defined as R ≡ 100 * (1 - Atreat /Acontr ).

Dissociated cell recordings

Whole-cell recordings employed standard techniques (Flores-Hernandez et al., 2002). Electrodes were pulled from Corning 8250 glass (A-M Systems, Inc, Carlsborg, WA) and heat polished prior to use. The internal solution consisted of (mM): 175 N-methyl-d-glucamine (NMDG), 40 HEPES, 2 MgCl2, 10 Ethylene glycol-bis(γ-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 12 phosphocreatine, 2 Na2ATP, 0.2 Na2GTP, 0.1 leupeptin, pH = 7.2-7.3 with H2SO4, 265-270 mOsm/L. The external solution consisted of (in mM): 127 NaCl, 20 CsCl, 5 BaCl2, 2 CaCl2, 12 glucose, 10 HEPES, 0.001 TTX, 0.02 glycine, pH = 7.3 with NaOH, 300-305 mOsm/L.

Recordings were obtained with an Axon Instruments 200A patch clamp amplifier and controlled with a Pentium clone running pClamp (v. 8.01) with a DigiData 1200 series interface (Axon Instruments, Foster City, CA). Electrode resistance was typically 2-4 MΩ in the bath. After seal rupture, series resistance (<20 MΩ) was compensated (70-90%) and periodically monitored. Recordings were made from pyramidal cells that had short (<75 μm) apical dendrites (Fig. 1B). Unless otherwise noted, the cell membrane potential was held at −80 mV.

Fig. 1. Acetylcholine and oxotremorine inhibit electrically-evoked NMDAR-mediated synaptic currents.

Fig. 1

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A: NMDAR-mediated synaptic currents were evoked by stimulating afferents in layer II/III with an extracellular electrode and recording them in an ACSF solution containing AMPAR and GABAAR and muscarinic receptor blockers at Vh = −40 mV. A pair pulse protocol was used to measure the PPR. The current was blocked by 100 μM APV. B: representative example of spiny neuron recorded in layer II/III. C and D: ACh (1 μM) inhibits postsynaptic INMDA without changing PPR. Traces are the average of 10 individual responses recorded every 20 seconds. E and F: the muscarinic agonist oxotremorine (10 μM) produces an effect similar to ACh, also without change in PPR

Drug application

Drugs were applied with a gravity-fed “two-pipe” system. The array of application capillaries -glued at approximately 800 μm from each other- was positioned a few hundred μm from the cell under study. Solution were applied by changing the position of the array with a DC drive system controlled by a SF-77B perfusion system (Warner Instruments, Hamden, CT) synchronized by pClamp. The solution changes were complete within less than 100 msec. Generally, NMDA (1-1000 μM) was applied for 3 sec every 20 sec. Drugs were prepared as concentrated stocks in deoxygenated water.

Results

Acetylcholine inhibits INMDA postsynaptically

In order to study the effect of cholinergic drugs on synaptic INMDA we recorded electrically-evoked NMDA currents (eEPSCs) in the presence of DNQX (10 μM), bicuculline (10 μM) or picrotoxin (100 μM), and atropine (10 μM) in the control solution, at a holding potential of −40 mV, in order to remove Mg2+ block. The NMDAR-mediated nature of the response was confirmed by complete block following the application of the NMDAR blocker amino-phosphonovaleric acid (APV, 100 μM, fig. 1A, n = 3). Neurons were injected with biocytin for post-hoc identification. A cortical layer II biocytin-injected reconstructed neuron is shown in fig. 1B.

Application of ACh (1 μM) or of the muscarinic agonist oxotremorine (oxo, 10 μM) decreased eEPSCs amplitude (R ≡ 100 * (1 - Atreat /Acontr ) = 36.4 ± 6 % and 37.8 ± 6.3 %, respectively, n = 8 each) without changing the pair pulse ratio (PPR) (PPR in control was 1.34 ± 0.13 vs. 1.43 ± 0.1 in ACh, n = 8, n.s, Wilcoxon's t-test; PPR = 1.22 ± 0.07 in control vs 1.25 ± 0.09 in oxo, n = 8, n.s, Wilcoxon's t-test). In fig. 1C, D, E and F, we show representative traces and PPR values illustrating the effect of ACh (C and D) and oxo (E and F). The invariance of the PPR following the application of ACh or oxo suggests, but does not prove, that the reduction of INMDA is due to a postsynaptic effect.

In order to test whether the cholinergic-induced INMDA inhibition was due to postsynaptic modulation we used a fast drug-application system to apply the prototypical agonist NMDA (100 μM, chemically-evoked or cEPSC) and/or other drugs to acutely-dissociated auditory cortical neurons. Application of NMDA produced large inward currents whose profile was unchanged during the application of ACSF (example in fig. 2A). On the contrary, the application of either ACh or oxo on top of the NMDA application resulted in a reversible decrease of the INMDA (R = 42.3 ± 2 %, n = 11, and R = 22.9 ± 1.4 %, n = 8, respectively; fig. 2B and C) whose onset and offset kinetics were only slightly slower than the drug dispensing speed of the system (τ = 1089 ± 28 ms, n = 11, example in the time expansion of fig. 2B). Mean ± s.e.m. of R, the reduction induced by ACh or oxo is shown in fig 2D.

Fig. 2. Acetylcholine and oxotremorine inhibit chemically evoked NMDAR-mediated currents.

Fig. 2

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A, B and C: Effect of application of ACSF, ACh or oxo on the current evoked by NMDA application in acutely dissociated neocortical neurons. Insert below B shows that the onset of the cholinergic block measured by an exponential curve with τ = 1089 ± 28 ms (n = 11). D: bars indicate mean ± s.e.m. of the reduction by ACh or oxo (n = 11 and 8, respectively). The asterisk (*) indicates a statistically significant reduction (P < 0.05, Student-t test).

The cholinergic inhibition of INMDA is not G-protein mediated

The resistance of cholinergic inhibition of INMDA to atropine suggested that muscarinic receptors were not responsible for the effect. In order to determine whether the activation of other GTP-dependent protein (G-protein) receptors was responsible for the INMDA inhibition we measured the effect of ACh on currents evoked by pressure application of NMDA using an intracellular solution containing the non-hydrolyzable analogue of guanosine-di-phosphate, GDPβS (500 μM) to lock the α-subunit of trimeric G-protein complexes in a permanently inactive state. In the presence of GDPβS in the pipette solution the application of a low concentration ACh (10 nM) or oxo (10 nM) still induced a reversible decrease in the cEPSC INMDA (examples in fig. 3A and B). In order to test the possible effect of endogenous ACh we determined the action of the ACh-esterase inhibitor physostigmine on the cEPSC. In the presence of GDPβS in the recording pipette, physostigmine reversibly decreased cEPSC amplitude, (10.6 ± 4 % in 10 μM, n = 4, and 28.4 ± 5% in 50 μM, n = 4, P < 0.05, t-test, examples in fig. 3C), raising the possibility that endogenous ACh reduces INMDA in a G-protein-independent fashion, similar to exogenously-applied ACh. Mean ± s.e.m. for the effects of ACh, oxo, or physostigmine on the cEPSC INMDA amplitudes are shown in fig. 3D.

Fig. 3. The cholinergic inhibition of (cEPSC) INMDA does not depend on G-proteins.

Fig. 3

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Recordings performed in the presence of GDPβS in the intracellular solution. A and B: ACh (10 nM) or oxo (10 nM) decrease the current evoked by the pressure application of NMDA. C: The cholinesterase inhibitor physostigmine (50 μM) also reduces cEPSC. D: Effect of different treatments on INMDA, normalized to control. Compare mean ± s.e.m. of the effects of cholinomimetics in the presence of GDPβS.

Nicotinic agonists and antagonists inhibit INMDA

To determine whether muscarinic drugs selectively inhibited INMDA, we tested the effect of the nicotine and tubocurarine - prototype nicotinic agonist and antagonist, respectively - on the amplitude of the cEPSC. Application of nicotine or of tubocurarine consistently inhibited INMDA in brain slices as well as in dissociated cells (fig. 4). In the presence of GDPβS, nicotine (10 μM) reversibly inhibited NMDAR-mediated cEPSC in brain slices (R = 58 ± 3%, n = 7) as well as in dissociated cells (R = 35.5 ± 8%, n = 5), as shown in the examples in fig. 4A and B. Similarly, tubocurarine, also in the presence of GDPβS, reversibly decreased cEPSC amplitude in brain slices (example in fig. 4C) as well as in dissociated cells (example in fig. 4D). Tubocurarine also inhibited electrically-evoked INMDA in brain slices (38.2 ± 6.6 %, n = 6, example in fig. 4E).

Fig. 4. Nicotine or tubocurarine inhibit INMDA.

Fig. 4

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A and B: Representative traces showing that nicotine (10 μM) reduces chemically-evoked INMDA in brain slices (A) and nicotine (10 nM) in dissociated neurons (B), recorded at Vh = −40 mV. C and D: Representative trace showing that tubocurarine (TB, 50 μM) inhibits electrically evoked synaptic INMDA (eEPSCs) in brain slices or dissociated cells (TB 50 nM), respectively. E: tubocurarine also reversibly inhibits eEPSC. F: Representative trace illustrating the effect of physostigmine on chemically induced INMDA in dissociated cells. G : Bar graphs report mean ± s.e.m. of the reduction in slices or dissociated cells, respectively. G: first and second column: reduction of chemically evoked INMDA by 10 μM nicotine or 50 μM tubocurarine, respectively, third column, reduction of synaptic INMDA by 50 μM tubocurarine. H: inhibition of INMDA by nicotine (10 nM) or tubocurarine (50 nM) and physostigmine (50 μM) in dissociated cells.

For ascertaining whether the inhibiting effect of physostigmine was due to its block of cholinesterase as suggested by the experiment in fig. 3C and D, or was rather a direct block of INMDA we also measured the effect of physostigmine bath-applications on cEPSC INMDA in dissociated cells. Unexpectedly, physostigmine significantly decreased INMDA (example in fig. 4F), indicative of a direct postsynaptic effect. Mean ± s.e.m. for the tubocurarine, nicotine and physostigmine inhibition of chemically-induced INMDA are summarized in the bar graphs in fig.4G and H for brain slices and dissociated cells, respectively.

In order to verify the effectiveness of intracellular dialysis, we used an intracellular solution with GTPγS (400 μM) - which like or GDPβS locks the α-subunit of trimeric G-protein complex, this time in a permanently active state - to test the effect of serotonin on the chemically evoked INMDA in recordings from cells of the medial prefrontal cortex, where 5-HT postsynaptically decreases NMDAR-mediated current through activation of the G-protein mediated 5-HT1A receptor (Yuen et al., 2005). As expected, we found that application of 5-HT (20 μM) reduced INMDA in control pipette solution (example in fig. 5A) but not in the presence of GTPγS (example in fig. 5B and 5C). On the contrary, tubocurarine (50 μM) still depressed INMDA even in the presence of GTPγS in the pipette solution (example in fig. 5C). The presence of GTPγS in the recording pipette produced similar results also in the auditory cortex, blocking any effect of 5-HT (R = 3 ± 2%, n.s., n = 4, data not shown), without changing the extent of the INMDA inhibition induced by tubocurarine (50 μM, R = 50 ± 4 %, n = 8, P < 0.05, paired t-Student).

Fig. 5. G-proteins are activated with GTPγS and tubocurarine still reduces INMDA.

Fig. 5

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A: 5-HT (20 μM) decreases chemically evoked INMDA in control, but not in the presence of GTPγS (400 μM, B). C: Tubocurarine (50 μM) but not 5-HT inhibit chemically-induced INMDA in the presence of GTPγS. D: mean ± s.e.m. of the reduction by 5-HT in control (n = 5), serotonin in GTPγS (n = 5), and tubocurarine (50 μM) in GTPγS (n = 4).

Mechanism of the cholinergic inhibition of INMDA

NMDARs receptors are subject to voltage-dependent block by neurotransmitters and modulators which carry multiple positive electric charges, and may block the channel pore in an activity- and voltage-dependent fashion (Cui et al., 2006). Furthermore, the NMDAR is subject to a block by Mg2+ ions which is relieved at depolarized potential (Mayer et al., 1984). We tested the possibility that the cholinergic inhibition was voltage-dependent by recording cEPSC INMDA at a depolarized holding potential (Vh) = + 40 mV. At this holding potential, cEPSC were recorded as outward currents, consistent with a reversal voltage for INMDA around 0 mV. Application of ACh, nicotine or tubocurarine inhibited INMDA to a similar extent measured at Vh = −40 mV (n = 4), suggesting that the reduction was not voltage-dependent (representative examples in fig. 6A, B, and C, respectively). In particular, ACh produced a dose-dependent response whose maximal effect was similar to that produced by either nicotine (10 nM - 10 μM, n = 6 each) or tubocurarine (50 μM, n = 4). Summary of these results are reported as mean ± s.e.m. in the graph bar in fig. 6D).

Fig. 6. The inhibition of INMDA is not voltage-dependent.

Fig. 6

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A, B, and C: Representative traces showing that ACh (10 nM or 10 μM), nicotine (10 nM or 10 μM), or tubocurarine (50 μM), respectively inhibit cEPSC INMDA in brain slices at a holding voltage Vh = + 40 mV. D: Mean ± s.e.m. of the reduction induced by ACh (10 nM, or 10 μM), nicotine (10 μM or 50 μM) or tubocurarine (50 μM).

Since all cholinergic drugs that we tested effectively inhibited INMDA we decided to test the effect of atropine (10 μM) on the amplitude of cEPSC in the presence of GTPγS (400 μM). Atropine applications did not change cEPSC amplitude (example in fig. 7A), while in the same recording conditions (GTPγS in the pipette solution) tubocurarine reliably induced a reversible reduction of INMDA, as shown in the example in fig. 7B.

Fig. 7. Effect of cholinergic antagonists.

Fig. 7

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Recordings in the presence of GTPγS (400 μM in the pipette solution A, Atropine (10 μM) was unable to reduce INMDA, while tubocurarine (50 μM, B) produced a completely reversible reduction in INMDA in two consecutive applications. C, Bar graphs report mean ± s.e.m. of the INMDA reduction by atropine, tubocurarine without glycine, or tubocurarine with glycine (n = 4).

The NR1 subunit of the NMDAR has a binding site for the aminoacid glycine and serine, whose presence is required for opening the channel following glutamate binding (Parsons et al., 1998). We considered the possibility that cholinergic inhibition of INMDA operated by modifying glycine or serine binding. To test this hypothesis we measured cEPSC INMDA in ACSF or after application of glycine (20 μM) to the extracellular solution, with a pipette solution containing GDPβS (500μM). In the presence of glycine, the tubocurarine-induced decrease in cEPSC INMDA amplitude more than doubled (mean ± s.e.m. in the bar graph in fig. 7C). An example of time course of the effects of tubocurarine on the cEPSC INMDA amplitude in the presence or in the absence of glycine is shown in the example in fig. 8A. For investigating in more depth tubocurarine-induced inhibition we obtained a dose-response of the inhibition by tubocurarine in the presence or in the absence of 20 μM glycine. We found that while in the nominal absence of glycine tubocurarine induced only a relatively modest decrease of the INMDA, in the presence of 20 μM glycine tubocurarine induced a reduction that was consistently larger throughout the dose-response curve, leading, for saturating doses of tubocurarine, to approximately the same current regardless of the presence or absence of glycine – around 40% of the maximal current in glycine (fig. 8B) -. A plot of the non normalized INMDA percentage reduction by tubocurarine (fig.8C) showed the greater potency of the tubocurarine reduction in the presence of glycine (maximum reduction by tubocurarine was 31 ± 2.5 % in control vs. 56 ± 7 % in 20 μM glycine). The complexity of the interaction between tubocurarine and glycine is illustrated by the change of the normalized dose response curve, showing a glycine-induced right-shift (fig. 7D, IC50tubocurarine(control) = 8 ± 1 nM vs. IC50 tubocurarine (control) = 98 ± 7 nM), with no change in the Hill coefficient. Small differences between INMDA reductions obtained with the same concentration of tubocurarine measured in GTPγS vs. GDPβS were not statistically significant. These results are compatible with the hypothesis of a non competitive mechanism of inhibition whereby tubocurarine displaces glycine from its binding site.

Fig. 8. Tubocurarine inhibits INMDA in a glycine dependent manner.

Fig. 8

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Recordings in the presence of GDPβS (500 μM) in the pipette solution A: Example of time course illustrating that tubocurarine (50 μM) exerts a modest reduction of INMDA in control. As expected, application of glycine (20 μM) greatly increases INMDA. Subsequent application of the same concentration of tubocurarine in the presence of glycine produces a much larger percent of inhibition, bringing INMDA to the minimum recorded current. All effects were completely reversible. B, C, and D: Dose-response of tubocurarine in the presence (red lines and symbols) or in the absence (black lines and symbols) of glycine (20 μM). B: Normalizing currents to the maximum current in the presence of glycine shows that saturating doses of tubocurarine bring INMDA to a minimal level independent on the presence of glycine (approximately 40% of the maximal current in glycine). C: Tubocurarine potency, measured as the maximum percentage reduction of the control current, is greatly increased in the presence of 20 μM glycine. D: the presence of glycine brings about large differences in the dose-response of INMDA to tubocurarine.

Discussion

Nicotinic agonists are suggested to inhibit INMDA through a non-competitive mechanism (O'Dell and Christensen, 1988). Our data confirm that nicotinic agonist inhibit NMDAR-mediated currents, and show for the first time that also the nicotinic antagonists tubocurarine, oxo, the endogenous neurotransmitter ACh, as well as the cholinesterase inhibitor physostigmine, all decrease INMDA in an atropine-insensitive manner, suggestive of a non-muscarinic mechanism. The unchanged PPR of the synaptic responses suggested a postsynaptic site of the effect, confirmed by the cholinergic reduction of currents evoked by the application of NMDA in brain slices as well as in dissociated neurons. The rapid kinetic of the reduction of INMDA induced by ACh and oxo in dissociated neurons suggested a direct interaction between the cholinergic drugs and the NMDAR.

Mechanism

Since several drugs and ions operate a voltage-dependent block of NMDAR-dependent currents, we tested whether cholinergics would inhibit INMDA at depolarized potentials to similar extent of the blockage in a hyperpolarized cell. No substantial differences were detected in the extent of the blockage at Vh = +40 mV vs. Vh = −40 mV, failing to demonstrate any voltage dependence of the inhibition.

Similar to the inhibition induced by ACh and oxo, the reduction in INMDA following nicotine and tubocurarine applications were not blocked by the presence of drugs preventing a large range of intracellular second-messenger cascades, suggesting that nicotinic and muscarinic substances alike might inhibit NMDAR-mediated currents using similar mechanisms.

We considered the possibility that cholinergic drugs interact at the glycine site by displacing the NMDAR co-agonist glycine. Because of its high effectiveness and reversibility as INMDA blocker, tubocurarine was selected as prototype inhibitor. While tubocurarine induced a large INMDA reduction in the presence of the NMDAR co-agonist glycine, the same substance induced only a modest but measurable, dose-dependent inhibition in nominal absence of glycine suggesting that tubocurarine might compete with this aminoacid displacing glycine from its binding site at the NR1 subunit. The specific mechanism of inhibition might differ for other cholinergic drugs.

Our data did not allow to assess whether INMDA is physiologically inhibited by ACh, as initially suggested by the inhibition of synaptic INMDA by the cholinesterase blocker physostigmine, since physostigmine itself inhibited INMDA also in dissociated cells, indicative of its direct interaction with the NMDAR. Our data do not completely rule out the possibility of a protein-protein interaction between the NMDAR and functional or non-functional ionotropic channels like those formed by the nicotinic α9 and/or α10 subunits, which possess a mixed muscarinic-nicotinic pharmacology (Baker et al., 2004; Lustig, 2006).

The cholinergic reduction of NMDARs is less surprising if we consider that NMDAR the “inverse” interaction (the block of nicotinic channels by NMDA antagonists) has already been reported in several studies (Aracava et al., 2005; Buisson and Bertrand, 1998a; Buisson and Bertrand, 1998b; Plazas et al., 2007) suggesting that at least nicotinic and NMDA ligands somehow overlap at least partly in their ligand affinities, although the specific mechanisms of their interactions might differ.

The present data would suggest an alternative interpretation to the finding that in the sensory neocortex, nicotine, but also carbachol, inhibit INMDA (Levy et al., 2006). Differences with our work might be due to the different cortical area (somatosensory vs. auditory) or different cortical layer (LV vs. LII/III), or shorter duration of the drug application. Our data also supply a simple explanation to a series of observations reporting that exposure to nicotine protects form NMDAR-mediated glutamate toxicity in a number of preparations (Ferchmin et al., 2003; Prendergast et al., 2001; Rezvani and Levin, 2003)

Physiological significance

Microdialysis studies have shown that the concentration of ACh measured in the presence of cholinesterase blockers is in the order of a fraction of micromolar (McIntyre et al., 2003). Even in the case that actual brain concentration of ACh would be two orders of magnitude lower, the significant reduction of INMDA observed for a concentration of ACh as low as 10 nM indicates that ACh could significantly inhibit tonically at least extrasynaptic NMDARs at physiological concentrations, while the transient reduction of synaptic NMDAR-mediated conductance would be possible following intense ACh release by cholinergic varicosities located in the neighborhood of glutamatergic synapses.

A cholinergic reduction of NMDARs could be an alternative or complementary mechanism for the induction of neocortical long-term depression which is associated to cholinergic modulation (Kirkwood et al., 1999). Considering that NMDARs and nicotinic receptors have high Ca2+ permeabilites, it is tempting to speculate that the effect of the increase in the physiologic concentrations of ACh in the neocortex would shift the distribution of neuronal Ca2+ entry from NMDAR-rich to nicotine receptors-rich neuronal regions, potentially introducing a parallel shift in the loci of synaptic plasticity.

Acknowledgments

This work has been supported by a NARSAD Young investigator grant to M.A. and the NIH grant NIH5R01DC005986 also to M.A.

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