Activation of muscarinic receptors modulates NMDA receptor-mediated responses in auditory cortex. &misc:Received: 7 March 1996 / Accepted: 14 August 1996 ...
Exp Brain Res (1997) 113:484–496
© Springer-Verlag 1997
R E S E A R C H A RT I C L E
&roles:V. Bessie Aramakis · Anita E. Bandrowski John H. Ashe
Activation of muscarinic receptors modulates NMDA receptor-mediated responses in auditory cortex
&misc:Received: 7 March 1996 / Accepted: 14 August 1996
&p.1:Abstract The present study examines the ability of muscarinic receptor activation to modulate glutamatergic responses in the in vitro rat auditory cortex. Whole-cell patch-clamp recordings were obtained from layer II-III pyramidal neurons and responses elicited by either stimulation of deep gray matter or iontophoretic application of glutamate receptor agonists. Iontophoresis of the muscarinic agonist acetyl-β-methylcholine (MCh) produced an atropine-sensitive reduction in the amplitude of glutamate-induced membrane depolarizations that was followed by a long-lasting (at least 20 min) response enhancement. Glutamate depolarizations were enhanced by MCh when elicited in the presence of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/ kainate receptor antagonists 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) or 2,3-diyhdroxy-6-nitro-7-sulfamoyl, benzo(F)quinoxaline (NBQX) but not the NMDA antagonists D-2-amino-5-phosphonovaleric acid (APV) or MK-801 hydrogen maleate. The magnitude of enhancement was voltage-dependent with the percentage increase greater at more depolarized membrane potentials. An involvement of NMDA receptors in these MCh-mediated effects was tested by using AMPA/kainate receptor antagonists to isolate the NMDA-mediated slow excitatory postsynaptic potential (EPSP) from other synaptic potentials. The slow EPSP and iontophoretic responses to NMDA were similarly modified by MCh, i.e., both being reduced during and enhanced (15–55 min) following MCh application. Cholinergic modulation of NMDA responses involves the engagement of G proteins, as enhancement was prevented by intracellular infusion with the nonhydrolyzable GDP analog guanosine-5′-O-(2thiodiphosphate) trilithium salt (GDPβS). GDPβS was without effect on the early MCh-induced response suppression. Our results suggest that acetylcholine, acting at muscarinic receptors, produces a long-lasting enhancement of NMDA-mediated neurotansmisson in auditory V. Bessie Aramakis · A.E. Bandrowski · J.H. Ashe ( ✉) Departments of Neuroscience and Psychology - 075, University of California, Riverside, CA 92521, USA; Fax: +1-909-787-3985&/fn-block:
cortex, and that this modulatory effect is dependent upon a G protein-mediated event. &kwd:Key words Glutamate · Muscarinic receptor · NMDA · Guanosine-5′-O-(2-thiodiphosphate) thrilithium salt · Response enhancement · Rat&bdy:
Introduction Acetylcholine (ACh) can modulate noncholinergic neurotransmission, by actions through muscarinic receptors, and consequently contribute to the regulation of neocortical excitability (for review see Aigner 1995). Regulation of glutamatergic transmission in neocortex is of particular importance, because glutamate is a major neurotransmitter involved in both thalamocortical and intracortical excitatory transmission (Thomson and Deuchars 1994; Salt et al. 1995). However, cholinergic modulation of glutamatergic transmission is complicated by the fact that glutamatergic actions are mediated by several glutamate receptors subtypes, including α-amino-3-hydroxy5-methyl-isoxazole-4-propionic acid/kainate (AMPA/ kainate), N-methyl-D-aspartate (NMDA), and metabotropic receptors (for review see Tsumoto 1990; Schoepp and Conn 1993). Furthermore, glutamatergic actions range from direct, fast mediated changes in ion conductance, predominantly through AMPA/kainte receptors, to slower modulatory/plastic actions that depend on NMDA and metabotropic receptors (reviewed in Daw et al. 1993; Schoepp and Conn 1993). The modulatory role of ACh in neocortical information processing has been implicated in many functions including arousal, attention, learning, and memory (Ashe and Weinberger 1991; Metherate et al. 1992; Nabeshima 1993). Of particular interest are findings that ACh, as well as other cholinergic agonists, modulate glutamatergic transmission by either presynaptic or postsynaptic mechanisms, or both (Yamamoto and Kawai 1967; Hounsgaard 1978; Segal 1989; Markram and Segal 1990; Lin and Phillis 1991; Krnjevic 1993; Cox et al. 1994;
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Kang 1995). These actions require specific subtypes of the muscarinic receptor with subsequent stimulation of intracellular second messenger systems (for review see Hosey 1992; Caulfield 1993). In rat auditory cortex, long-lasting postsynaptic enhancement of glutamate depolarizations is mediated by the M1 and/or M3, rather than the M2, muscarinic receptor subtype (Cox et al. 1994). This muscarinic action of ACh is essentially mimicked by activation of protein kinase C by phorbol 12,13dibutyrate (4-β-PDBu) and is attenuated by the kinase antagonist 1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H7). Thus, there is strong evidence that ACh can modulate glutamatergic neurotransmission, and in rat auditory neocortex there is differential involvement of muscarinic receptor subtypes. However, an important unanswered question is the susceptibility of neocortical glutamate receptor subtypes to modulation by ACh. In the present study, we used whole-cell patch-clamp recording in rat auditory cortex to examine the susceptibility of NMDA-mediated neurotransmission to cholinergic modulation. Our results demonstrate that the muscarinic agonist acetyl-β-methylcholine (MCh) produces a biphasic change in NMDA receptor-mediated auditory cortical responses. This effect consists of an initial reduction, followed by a lasting enhancement. This latter effect is in agreement with previous findings from hippocampal pyramidal neurons (Markram and Segal 1990). Further, we provide evidence that long-lasting enhancement in auditory cortex is postsynaptically dependent upon a G protein-coupled mechanism (see Markram and Segal 1992 for hippocampus). Part of this work has appeared in abstract form (Aramakis et al. 1994).
Materials and methods Tissue preparation and maintenance The methods used for brain slice preparation and maintenance are similar to those described previously (Cox et al. 1992, 1994; Metherate and Ashe 1994). Male Sprague-Dawley rats (3–5 weeks old and weighing approximately 80–120 g) were anesthetized with pentobarbital sodium (Nembutal, 25 mg/kg). Following decapitation, bains were rapidly removed and placed in cold artificial cerebrospinal fluid (ACSF) containing: NaCl 125.0 mM; KCl 2.5 mM; NaHCO3 25.0 mM; KH2PO4 1.25 mM; MgSO4 1.2 mM; CaCl2 2.5 mM glucose 10.0 mM; and bubbled with 95% O2, 5% CO2, to a pH of approximately 7.4. Osmolarity of the bathing solution was 298–302 mosmol/kg. Coronal slices, 350–400 µm in thickness, were obtained from auditory neocortex (Sally and Kelly 1988; Cox et al. 1992) with the use of a vibrating tissue slicer, and placed in either an interface-type recording chamber or a holding flask. Chamber slices were superfused with ACSF at a rate of 1.5–2.0 ml/min., and warmed (32° C), moistened gas (95% O2, 5% CO2) was continuously directed over their surface. Until transferred to the recording chamber, slices in the holding flask were maintained at room temperature and continuously oxygenated. For initial chamber slices, data collection began 2 h after entering the chamber, and after 1 h for slices transferred from the holding flask.
Electrophysiological recording and stimulation Micropipettes for whole-cell recordings (WCR) were made from glass capillary tubes, using a horizontal microelectrode puller, and had tip diameters of approximately 2.5 µm and d.c. resistances of 4–6 MΩ. These pipettes were filled with 120.0 mM potassium gluconate, 2 mM MgCl2, 2 mM CaCl2, 10 mM ethyleneglycol-bis(β-amino ethyl ether) N,N′-tetra-acetic acid (EGTA), 10 mM N-2hydroxyethylpiperazine-N′-2 ethanesulphonic acid (HEPES), and adjusted to a pH of 7.3 with KOH (1.0 M). In some experiments, 200 µM guanosine-5′-O-(2-thiodiphosphate) trilithium salt (GDPβS) was added to the WCR solution. Final osmolality of the WCR solution was adjusted to 277–282 mosmol/kg. Slices were transilluminated and viewed through a dissecting microscope. Recordings were made from neurons located 300–450 µm from the pia surface corresponding to layer II–III of rat auditory neocortex (Roger and Arnault 1989). A bipolar stimulating electrode (insulated stainless steel, 200 µm tip diameter) was placed either in deep gray matter (1000–1500 µm from the pia) or in underlying white matter, in line with the recording electrode (i.e., in the same cortical “column”). Stimulation consisted of 0.1ms-duration, 5- to 100-µA monophasic constant-current pulses. Whole-cell patch recordings were obtained as previously described (Blanton et al. 1989; Metherate and Ashe 1994, 1995a). Following establishment of the whole-cell configuration (1–2.5 GΩ seal, 6–25 MΩ series resistance), compensation for series resistance was accomplished by using the discontinuous current clamp (switching) mode of the amplifier (Axoclamp 2A; Axon Instruments), to measure the membrane response to steady hyperpolarizing current (0.3–0.5 nA). Switching frequencies were 5–8 kHz and headstage voltage was monitored on a separate oscilloscope to ensure adequate electrode settling time and optimal capacitance compensation. After changing to the continuous currentclamp (bridge) mode, the disparity between the membrane potential (Vm) in continuous versus discontinuous modes was noted and compensated by adjusting series resistance compensation (Metherate and Ashe 1994). Compensation was adjusted at regular intervals, and series resistance generally ranged from 6 to 25 MΩ initially, but could increase to 20–40 MΩ following several hours of recording. Vm was monitored using an oscilloscope and chart recorder, and computer software (AXODATA; Axon Instruments) was programmed for stimulus delivery and data collection. Recordings were digitized at a sampling rate of 2 kHz (MIO-16; National Instruments), then stored on computer (Macintosh Quadra 900) and videotape (bandpass 0–5 kHz; A.R. Vetter) for further analysis. Cell staining In a number of experiments, electrodes contained biocytin (0.2–0.5%), and commonly used tissue processing procedures were used for subsequent visualization of neurons (Horikawa and Armstrong 1988; Kita and Armstrong 1991). Slices were fixed in 4% paraformaldehyde for at least 24 h, resectioned at 50 µm on a freezing microtome, and processed using the avidin-biotin-peroxidase method (ABC Kit; Vector Laboratories). Biocytin-filled cells were reconstructed using camera lucida drawings of the serial sections. Pharmacological agents and application The following pharmacological agents were used: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3dione (DNQX; from Tocris Neuramin); 2,3-dihydroxy-6-nitro-sulfamoyl benzo(F)quinoxaline (NBQX; from Novo Nordisk); NMDA, MCh, D-2-amino-5-phosphonovaleric acid (APV), (+)MK-801 hydrogen maleate (MK-801) and GDPβS (from Research Biochemicals); L-glutamic acid monosodium salt (glutamate), and atropine sulfate (from Sigma Chemicals). Agonist delivery, by iontophoresis, was with a three-barrel micropipette that contained the pharmacological agents of interest in
486 two barrels and NaCl (1.0 M) in the third barrel for current balance. Drugs used were glutamate (1 M), NMDA (50–100 mM), and MCh (1 M), all dissolved in distilled water. Typical placement of the iontophoretic micropipette was about 50 µm lateral to the recording electrode, in the same cortical layer. Ejection currents ranged from 10 to 150 nA, with holding currents ranging from 5 to 20 nA. Receptor antagonists, i.e., APV, MK-801, CNQX, NBQX, or atropine were bath-applied for at least 20 min before initiating the experiment (Fig. 1B, ii). Antagonists were prepared as a concentrated stock solution in distilled H 2O and diluted to their final concentration with ACSF. Preparation of WCR solution containing GDPβS was similar to that described by Andrade (1994). Aliquots (1 ml) of 1 mM GDPβS, dissolved in distilled water, were stored at −28° C. When needed, 4 ml of WCR solution were mixed with 1 ml of GDPβS (1 mM), to yield a final concentration of 200 µM GDPβS. GDPβS was introduced into cells by passive diffusion from the patch pipette. The basic design of these experiments is illustrated in Fig. 1B. Repetitive iontophoretic ejection of glutamate agonists was as follows: 10- or 20-s pulses at a frequency of 1/60 s for glutamate, and 5-s pulses at a frequency of either 1/90 s or 1/120 s for NMDA. Repetitive stimulation with glutamate was for at least 5 min, and for NMDA at least 10 min before MCh iontophoresis. Both glutamate agonists were delivered during MCh application (3–6 min) and were continued for at least 15 min after MCh application. Data analysis Amplitudes of synaptic potentials and agonist-induced responses were measured from baseline to peak. For both of these, changes are expressed as “percentage change from control amplitude.” Analysis of the variation in control amplitudes of glutamate depolarizations, over periods of 15 min or longer, yielded a 95% confidence interval, i.e., P≤0.05, at a 29% change in response amplitude (n=9). Consequently, response suppression was defined as at least a 29% decrease from the predrug mean amplitude and response enhancement defined as at least a 29% increase from the mean predrug amplitude. Means are presented as the mean±1 SEM. Differences between means were evaluated for statistical significance using the t-test for independent samples or, where appropriate, the t-test for paired samples. Correlation coefficients were calculated using the product-moment method. Evaluation of effects as a function of time were made using analysis of variance (ANOVA). All differences were considered statistically significant if the probability of occurrence by chance was 0.05 or less. Unless indicated otherwise, probability values in the text refer to results from t-tests.
Results Recordings were made from 126 neurons located between 300 and 450 µm from the pia, corresponding approximately to layer II-III of rat auditory neocortex (Sally and Kelly 1988; Roger and Arnault 1989). Mean resting Vm was −69.0 mV (±0.6 mV, n=126), with mean input resistance (Ri) of 87.3±4.3 MΩ (n=89). Measurement of Ri immediately followed series resistance compensation, i.e., within 5–10 min of establishing the whole-cell configuration. Cells included in this report are likely pyramidal neurons as judged from examination of biocytin-filled neurons (n=55) and standard electrophysiological criteria, i.e., spike duration, spike frequency accommodation, and spike rise/decay time (Connors et al. 1982; McCormick
et al. 1985). Morphological characteristics of neurons filled with biocytin included pyramidal-shaped cell bodies with a large apical dendrite extending toward the pia (Fig. 1A). Fast-spiking cells (Connors et al. 1982; McCormick et al. 1985) were only occasionally encountered and these are not included in the present report. Agonist effects Glutamate-induced responses Iontophoretically applied glutamate (10 s or 20 s) produced membrane depolarizations often of sufficient amplitude to elicit discharge of multiple action potentials. Thus, in order to study glutamate depolarizations and their modification by cholinergic actions without cell discharge, ejection current was adjusted (5–100 nA) to produce only subthreshold membrane depolarizations. These responses had onset latencies that ranged from 0.16–1.1 s and reached a maintained peak amplitude of 1–14 mV within 2–3 s (Figs. 1C, D, 2A). For the most part, responses rapidly decayed back to baseline levels within ~1-3 s following glutamate ejection. Occasionally there was a small undershoot below baseline Vm , but this was not further analyzed because of its brief duration and small amplitude. For any given neuron, glutamate responses were relatively constant in amplitude and duration when elicited at a frequency of 1 per 1–2 min (Figs. 1C, D, 2A), and they were blocked by the combination of AMPA/kainate and NMDA receptor antagonists (n=5; data not shown). Ejection of NaCl using comparable ejection currents had no effect on Vm (see Fig. 2A, i). MCh-induced responses MCh (30–150 nA for 180 s) produced a slowly developing and dose-dependent membrane depolarization (onset 14.5±6.2 s) with a peak amplitude of 4.3±0.4 mV and duration of 232.9±20.3 s (n=10; Fig. 1C, i and ii; see also Cox et al. 1994). For 33 of 58 neurons in which Vm was held at the resting level with constant current, MCh increased Ri by an average of 29.4±5.3% (Fig. 1C, i, ii). Ri in the remaining 25 cells was either decreased (16.7±2.5%, n=11) or did not change (n=14). Atropine (1 µM) blocked both the MCh-induced membrane depolarization and change in Ri (Fig. 1C; see also Cox et al. 1994). MCh modulation of glutamate-induced responses The amplitude of glutamate-induced depolarizations recorded from neurons exposed to MCh differed over the course of an experiment (F19,215=2.0, P0.05; Fig. 1C, D). Moreover, glutamate responses were modified differently in
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Fig. 1 A Camera lucida drawings of characteristic layer II-III pyramidal neurons filled with 0.2% biocytin (each has been reconstructed from two 50-µm serial sections). Scale bar 40 µm. B Diagrammatic representation of the basic experimental design. i. Iontophoretic ejection of either glutamate (10 s or 20 s) or NMDA (5 s) was adjusted to produce subthreshold membrane depolarizations. Ejections were repeated at a frequency of 1/60 s (glutamate) or 1/90–120 s (NMDA), before during, and after acetyl-β-methylcholine (MCh) iontophoretic ejection (3–6 min; black bar). ii. When glutamate antagonists also were used [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2,3-dihydroxy-6-nitro-7-sulfanoyl benzo(F)quinoxaline (NBQX), D-2-amino-5-phosphonovaleric acid (APV), or (+)-MK-801 hydrogen maleate (MK-801)], they were bath-applied throughout the protocol, and for at least 20 min (stippled bar) prior to initiating the sequence described in i. C MCh-enhancement of glutamate responses is dose-dependent and atropine-sensitive i. Glutamate (Glu) ejection (−21 nA for 10 s) produced subtrheshold membrane depolarizations of approximately 2.5 mV. These responses were elicited once per minute, and hyperpolarizing current pulses (0.10 nA, 40 ms) were given at a rate of 1/5 to monitor input resistance (Ri). MCh application (50 nA for 4 min; black bar) resulted in depolarization of approximately 6 mV, a small increase in Ri, and smaller, superimposed glutamate responses. Current clamp of membrane potential (Vm) to resting level, during MCh ejection (at arrowhead), revealed that glutamate responses were decreased by about 11%, and Ri increased by about 6%. MCh-induced depolarization recovered to baseline within 4 min following ejection; however, glutamate responses attained a peak enhancement of about 50% and remained elevated for at least 18 min post-MCh application. ii. After partial recovery
(i), an additional, but stronger application of MCh (75 nA for 4 min) produced larger membrane depolarization, larger increase in Ri, and greater reduction of glutamate response. Vm was clamped to resting level, during MCh application (at arrowhead). Following MCh application, glutamate responses reached a peak enhancement of about 76%, accompanied by a 47%, increase in Ri. MCh depolarization lasted about 6 min and enhanced glutamate responses for at least 20 min post-MCh application. iii. The effects of MCh on Vm, Ri, and glutamate responses were blocked by superfusion with atropine (1 µM). D Membrane responses to repeated glutamate ejection. Plotted is the mean percentage change (±SE) of Vm from control level (mean glutamate response for the first 3 min). In the presence of MCh (30–150 nA; black bar on the abscissa), there was a decrease in glutamate response amplitude (ACSF/MCh, closed circles; n=12, P
