| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201; 2 Departamento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ 21941-590, Brazil
Submitted 6 March 2003; accepted in final form 28 March 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
7* and 3
4* nicotinic acetylcholine receptors (nAChRs), respectively, present in glutamatergic neurons. This study examines how nicotinic AMPA, and NMDA receptor nAChR activities are integrated to regulate the excitability of CA1 stratum radiatum (SR) interneurons in rat hippocampal slices. At resting membrane potentials and in the presence of extracellular Mg2+ (1 mM), nicotinic agonists triggered in SR interneurons excitatory postsynaptic currents (EPSCs) that had two components: one mediated by AMPA receptors, and the other by NMDA receptors. As previously shown, nicotinic agonist–triggered EPSCs resulted from glutamate released by activation of 34* nAChRs in glutamatergic neurons/fibers synapsing directly onto the neurons under study. The finding that CNQX caused more inhibition of nicotinic agonist–triggered EPSCs than expected from the blockade of postsynaptic AMPA receptors indicated that this nicotinic response also depended on the AMPA receptor activity in the glutamatergic neurons synapsing onto the interneuron under study. Nicotinic agonists always triggered action potentials in CA1 SR interneurons. In most interneurons, these action potentials resulted from activation of somatodendritic AMPA receptors and 7* nAChRs. In interneurons expressing somatodendritic 42* nAChRs, activation of these receptors caused sufficient membrane depolarization to remove the Mg2+-induced block of somatodendritic NMDA receptors; in these neurons, nicotinic agonist–triggered action potentials were partially dependent on NMDA receptor activation. Removing extracellular Mg2+ or clamping the neuron at positive membrane potentials revealed the existence of a tonic NMDA current in SR interneurons that was unaffected by nAChR activation or inhibition. Thus integration of the activities of nAChRs, NMDA, and AMPA receptors in different compartments of CA1 neurons contributes to the excitability of CA1 SR interneurons. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
; Sargsyan et al. 2001) are intimately associated with learning (Huerta and Lisman 1993, 1995). Although changes in these rhythms (Bland and Colom 1988; Cobb et al. 1999; Yamamoto 1998) could underlie the learning improvement induced by nicotinic agonists (Levin 2002), the cellular mechanisms by which nicotinic acetylcholine receptors (nAChRs) regulate the activity of hippocampal networks are poorly understood.
The excitability of a given CA1 interneuron is driven by glutamate inputs (McBain et al. 1999) and is regulated by various neurotransmitter receptors, including the nAChRs. Both 7* 1 and 42* nAChRs are present in the somatodendritic and preterminal regions of CA1 interneurons in the stratum oriens (SO), stratum radiatum (SR), and stratum lacunosum moleculare (SLM) (Alkondon and Albuquerque 2001; Alkondon et al. 1999, 2000; Frazier et al. 1998a; Ji and Dani 2000; Jones and Yakel 1997; McQuiston and Madison 1999; Sudweeks and Yakel 2000).
Nicotinic–glutamatergic interactions have been best characterized at the 7* nAChR level. Activation of 7* nAChRs triggers action potential-independent glutamate release from axon terminals (Alkondon et al. 1996; Girod et al. 2000; Gray et al. 1996; Ji et al. 2001; Mansvelder and McGehee 2000; Mansvelder et al. 2002; McGehee et al. 1995). Further, in rats and mice, 7* nAChR activation enhances field stimulation-evoked glutamate currents, leading sometimes to long-term potentiation (Aramakis and Metherate 1998; Ji et al. 2001; Mansvelder and McGehee 2000; Santos et al. 2002). Only a few reports have indicated that non-7 nAChRs regulate glutamatergic synaptic transmission (Gil et al. 1997; Guo et al. 1998; Vidal and Changeux 1993). Although modulating glutamatergic inputs to hippocampal interneurons can have serious consequences to the hippocampal function, only limited information is available on the modulatory role of nAChRs on glutamatergic activity to the interneurons (Alkondon and Albuquerque 2002).
Considering that 1) interneurons are crucial for sustaining the excitability and firing pattern of principal neurons (Freund and Buzsaki 1996; McBain et al. 1999) and are thus critical for pacing rhythmic oscillations that set the window for learning (Huerta and Lisman 1993; McMahon et al. 1998), 2) nicotinic–glutamatergic interactions are important for synaptic plasticity underlying cognitive functions (Ji et al. 2001; Mansvelder and McGehee 2000), and 3) NMDA mechanisms account for the actions of nicotine in many brain regions (Aramakis and Metherate 1998; Mansvelder and McGehee 2000; Schilström et al. 1998; Shoaib et al. 1994), it becomes essential to define how the nicotinic cholinergic system controls the glutamatergic drive to the interneurons. The primary goals of this study were 1) to identify the glutamatergic receptors activated in CA1 SR interneurons in response to nicotinic agonists, 2) to determine the mechanisms by which nicotinic agonists regulate glutamatergic inputs to these interneurons, 3) to evaluate how nicotinic ligands modulate the excitability of CA1 SR interneurons, and 4) to investigate whether glutamatergic receptors are tonically active in CA1 interneurons and, if so, whether nicotinic ligands affect this tonic activity. The results presented herein demonstrate that nicotinic agonists increase the excitability of CA1 SR interneurons by NMDA receptor-dependent and -independent mechanisms. A preliminary account of this work was presented at the 32nd Annual Meeting of the Society for Neuroscience (Program number 242.2, 2002).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Slices of 250-µm thickness were obtained from the hippocampus of 16- to 18-day-old Sprague–Dawley rats according to the procedure described earlier (Alkondon and Albuquerque 2001). Animal care and handling were done strictly in accordance with the guidelines set forth by the Animal Care Committee of the University of Maryland, Baltimore. Slices were stored at room temperature in artificial cerebrospinal fluid (ACSF), which was bubbled with 95% O2-5% CO2 and composed of (in mM): NaCl, 125; NaHCO3, 25; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 2, MgCl2, 1; and glucose, 25. SR interneurons in the CA1 field of the slices were visualized by means of infrared-assisted videomicroscopy. In most experiments, biocytin labeling was also used to identify the neurons morphologically (Alkondon and Albuquerque 2001).
Electrophysiological recordings
Excitatory postsynaptic currents (EPSCs) and nicotinic currents were recorded from the soma of CA1 SR interneurons according to the standard whole cell patch-clamp technique (Hamill et al. 1981), using an LM-EPC7 amplifier (List Electronic, Darmstadt, Germany). Agonists were applied to the slices by a U-tube, and antagonists were applied by both U-tube and bath perfusion (Alkondon and Albuquerque 2001). Some test antagonists were applied only by bath perfusion. The U-tube had a pore diameter of 300 µm, and was positioned about 200 µm above the surface of the slice. This distance in addition to the fact that negative pressure was applied to the output of the U-tube to prevent agonist leakage caused some delay in the agonist delivery to the neurons. For this reason, agonist pulses below 1.5 s were found to be ineffective in producing any responses. Thus the start of agonist pulses shown in the figures represents the activation of the valve rather than the actual beginning of agonist outflow. Signals were filtered at 3 kHz and either recorded on a videotape recorder for later analysis or directly sampled by a microcomputer using the pCLAMP6 program (Axon Instruments, Foster City, CA). Neurons were superfused with ACSF at 2 ml/min. Nominally Mg+2-free ACSF was used in some experiments. Atropine (1 µM) and bicuculline (10 µM) were added to the ACSF to block muscarinic and GABAA receptors, respectively. In some experiments, methyllycaconitine (MLA, 10 nM) was also included in the ACSF to block 7* nAChR activity. Patch pipettes were pulled from a borosilicate glass capillary (1.2-mm OD), and when filled with internal solution had resistances between 3 and 6M
. The series resistance ranged from 8 to 20 M.At –68 mV, the leak current was generally between 50 and 150 pA, and when it exceeded 200 pA, the data were not included in the analysis. For most voltage-clamp recordings, the internal solution contained 0.5% biocytin in addition to (in mM): EGTA, 10; HEPES, 10; Cs-methanesulfonate, 130; CsCl, 10; MgCl2, 2; and lidocaine N-ethyl bromide (QX-314), 5 (pH adjusted to 7.3 with CsOH; 340 mOsm). Membrane potentials were corrected for liquid junction potentials. In some neurons, recordings of fast current transients (the counterparts of action potentials under voltage clamp) were initially obtained under cell-attached configuration before entering into the whole cell configuration. In so doing, the ability of nicotinic agonists to initiate action potential firing in the interneurons could be assessed without perturbing the intracellular environment. All experiments were carried out at room temperature (20–22°C).
Data analysis
Peak amplitudes and decay-time constants of EPSCs mediated by NMDA and AMPA receptors were analyzed using the CDR (Dempster 1989) or the pCLAMP6 software. The net charge of the AMPA and NMDA component of EPSCs was calculated using the pCLAMP6 software. Tonic NMDA currents were measured by the SD of the baseline current recorded from neurons under whole cell configuration. Segments of data (1-s duration each) containing no phasic events were selected and the SD determined using the pCLAMP6 software. For each experimental condition, 4–5 segments of recordings obtained from a given neuron were analyzed and the average value was used. Results are presented as means ± SE, and compared for their statistical significance by either paired or unpaired Student's t-test.
Drugs and toxins used
ACh chloride, DL-2-amino-5-phosphonovaleric acid (APV), (–)-bicuculline methiodide, choline chloride, cytisine, DMPP, (–)-nicotine hydrogen tartrate, tetrodotoxin (TTX), QX-314, and atropine sulfate were obtained from Sigma Chemical (St. Louis, MO). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was purchased from Research Biochemicals International (Natick, MA). MLA citrate was a gift from Professor M. H. Benn (Dept. of Chemistry, University of Calgary, Alberta, Canada). Dihydro--erythroidine (DHE)·HBr and (±)-mecamylamine·HCl were gifts from Merck, Sharp & Dohme Research Laboratories (Rahway, NJ). Stock solutions of all drugs were made in distilled water.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Morphological features of the CA1 SR interneurons studied electrophysiologically
After the electrophysiological recordings were obtained, post hoc reconstruction of the image of biocytin-filled interneurons (n = 55) revealed that their cell bodies were located in the CA1 SR and their axons ramified densely within the CA1 SR. Most interneurons (n = 36) had their axon arborization projecting into the SO. The dendrites of the majority of the CA1 SR interneurons (n = 41) were mostly confined to the CA1 SR. Only a few interneurons had their dendrites extended to the CA1 SLM and/or SO. The morphological characteristics of the SR neurons studied herein were distinct from those described earlier for CA1 SR giant cells (Gulyás et al. 1998).
Activation of nAChRs triggers phasic glutamatergic events mediated by both AMPA and NMDA receptors in CA1 SR interneurons
In the continuous presence of extracellular Mg2+ (1 mM), U-tube application (12 s) of DMPP (0.1 mM) to the area surrounding and including CA1 SR interneurons voltage clamped at –60 mV (i.e., near to their resting membrane potential) triggered bursts of inward going EPSCs (Fig. 1Aa). These events occurred both during the application of the agonist and up to another 20 s after the end of a 12-s pulse. These EPSCs displayed rapidly decaying as well as slowly decaying current components with corresponding mean 
-decay values of 2.1 and 86 ms (see expanded single event marked by an asterisk in Fig. 1Aa). The magnitude of both EPSC components triggered by DMPP decreased substantially when agonist pulses were applied at intervals 
5 min. Throughout the study, all agonist pulses were applied at intervals of 
9 min.
|
The slow and fast components of the DMPP-triggered EPSCs were reversibly blocked by a 10-min perfusion of the hippocampal slices with ACSF containing APV (50 µM) or CNQX (10 µM), respectively (Fig. 1Ac–e). These results indicated that the slow and fast EPSC components were mediated by NMDA and AMPA receptors, respectively.
Depolarization of CA1 SR interneurons from –60 to –40 mV decreased the total peak amplitude, increased the net charge and prolonged the decay phase of the slow component, and reduced the net charge of the fast component of the DMPP-triggered EPSCs (Fig. 1, Ab and B). Analyses of the recordings shown in Fig. 1A revealed that in that neuron, the average charge carried by the DMPP-triggered EPSCs at –60 mV was 2.11 pC per event, of which only 0.6 pC (i.e., approximately 28%) was contributed by AMPA EPSCs. In the same neuron, the average charge carried by DMPP-evoked EPSCs at –40 mV was 3.36 pC per event, of which 0.4 pC (i.e., approximately 12%) were contributed by AMPA EPSCs. The voltage-dependent changes in the values of -decay and net charge of each component of the DMPP-triggered EPSCs recorded from several neurons are shown in Table 1. The increased contribution of the NMDA component to the total charge carried by DMPP-triggered EPSCs observed with membrane depolarization from –60 to –40 mV (Fig. 1B) could be accounted for by the enhanced activity of NMDA receptors observed between –60 and –30 mV (Fig. 1C) most likely as a consequence of release of Mg2+-induced block of these receptors.
|
Even though at –60 mV, AMPA receptor-mediated EPSCs contributed to approximately 12% of the total charge carried by DMPP-triggered EPSCs in several neurons in the presence of extracellular Mg2+ (1 mM; Table 1), perfusion of the hippocampal slices with ACSF containing CNQX (10 µM) caused a substantial reduction of the total charge of the DMPP-evoked EPSCs (Fig. 1Ae). Likewise, in experiments carried out with nominally Mg2+-free ACSF, CNQX (10 µM) reduced by approximately 48% the total charge carried by DMPP-triggered EPSCs (Fig. 1D). The finding that CNQX-induced blockade of the DMPP-induced EPSCs was larger than it would have been expected from the inhibition of the postsynaptic AMPA receptors, it can be concluded that part of the DMPP-triggered EPSCs result from the indirect activation of AMPA receptors that regulate glutamate inputs to the interneurons under study.
To determine how the contribution of the NMDA component of DMPP-triggered EPSCs changes at any given membrane potential, a correlational analysis of peak amplitude and net charge of DMPP-evoked EPSCs was performed for results obtained from each interneuron voltage-clamped at –60 mV. The magnitude of the slow EPSC component depended on the size of the total EPSC (see individual events shown in Fig. 2A). The net charge of the DMPP-triggered EPSCs increased linearly with their peak amplitude (Fig. 2A) even when the voltage remained constant; the correlation coefficient was 0.96. Results obtained from similar experiments resulted in correlation coefficients ranging from 0.47 to 0.99 (mean ± SE = 0.76 ± 0.08; n = 8 neurons). Large, summated NMDA EPSCs could be easily detected in recordings obtained from interneurons that responded to DMPP with bursts of EPSCs (Fig. 2B). Analysis of these recordings revealed that the net charge of DMPP-triggered EPSCs increased as a function of the number of EPSCs inside the bursts. The correlation coefficient of the plot of net charge versus number of events inside the bursts of DMPP-evoked EPSCs in five neurons was 0.79 (Fig. 2B). These findings indicate that an increase in the quantal release of glutamate by nicotinic agonists enhances the probability of activation of NMDA receptors in the SR interneurons.
|
Receptor antagonists were used to help identify the origin and nature of the DMPP-triggered NMDA EPSCs in CA1 SR interneurons. To release the Mg2+-induced block of NMDA receptors, EPSCs were recorded in response to 12-s pulses of DMPP from interneurons voltage-clamped at +40 mV. The majority of the DMPP-induced EPSCs occurred as bursts of overlapping events that sometimes resulted in large, summated EPSCs (see Fig. 3, A and B). At +40 mV, all isolated EPSCs evoked by DMPP had -decay of 310 ± 25 ms (n = 25 neurons). Perfusion of the hippocampal slices with APV (50 µM)-containing ACSF decreased by approximately 89% the net charge of the DMPP-induced EPSCs (Fig. 3, A and D). At the positive membrane potential, the slow -decay and the sensitivity of the DMPP-triggered events to block by APV indicated that they were mediated primarily by NMDA receptors. A few fast-decaying EPSCs were recorded from CA1 SR interneurons in response to DMPP in the presence of APV (Fig. 3A). These events were sensitive to block by CNQX (data not shown) and were thus mediated by AMPA receptors. Fast-decaying, AMPA-mediated EPSCs contributed only about 1% of the total charge of the EPSCs triggered by DMPP at +40 mV (see Table 1).
|
Similarly to its inhibitory effect on DMPP-triggered EPSCs at –60 mV, CNQX (10 µM) alone caused substantial inhibition of DMPP-induced EPSCs recorded from the interneurons at +40 mV (Fig. 3, B and D); it decreased by approximately 55% the net charge of DMPP-triggered EPSCs. The ability of DMPP to trigger NMDA EPSCs was not affected after a 10-min perfusion of the hippocampal slices with ACSF containing the 7 nAChR antagonist MLA (10 nM; Fig. 3D). In contrast, after perfusion of the hippocampal slices with ACSF containing the 34 nAChR-preferring antagonist mecamylamine (Papke et al. 2001; Xiao et al. 1998), DMPP induced fewer or no EPSCs. At 1 µM (Fig. 3, C and D) mecamylamine decreased by approximately 80% the net charge of DMPP-triggered EPSCs. Further, at 0.1 and 0.3 µM, mecamylamine produced about 48 and 60% inhibition, respectively, of the net charge of DMPP-evoked NMDA EPSCs, suggesting an IC50 value in this concentration range. These results, in addition to the finding that TTX (0.5 µM) prevented DMPP from triggering NMDA EPSCs (Fig. 3D), suggested that activation of 34* nAChRs in preterminal/somatodendritic regions of glutamatergic neurons synapsing onto the neurons under study facilitates the release of glutamate, which in turn activates NMDA receptors in the interneurons. Further, the inhibitory action of CNQX on DMPP-induced EPSCs supported the notion that nAChR-induced excitation of glutamate neurons is amplified by indirect activation of AMPA receptors.
Nicotinic agonists aid in the discrimination of subtypes of native and reconstituted nAChRs subserving a given nicotinic response (Albuquerque et al. 1997; Alkondon and Albuquerque 2002; Kuryatov et al. 2000; Luetje and Patrick 1991; Pereira et al. 2002; Quick et al. 1999). Further characterization of the pharmacological profile of the nAChR subtype subserving nicotinic agonist-induced EPSCs was made possible by the use of choline, DMPP, nicotine, and cytisine. Choline acts as full agonist on rat 7 nAChRs and as partial agonist on rat 9, 44, and 34 nAChRs (reviewed in Pereira et al. 2002); cytisine acts as a very weak agonist on 2-containing nAChRs (Buisson et al. 1996; Nelson et al. 2001; Papke and Heinemann 1994; see also Fig. 3G); and DMPP, nicotine, and cytisine activate rat 34 nAChRs with similar potency (reviewed in Vizi and Lendvai 1999).
Choline (10 mM; 12-s pulses) induced very few EPSCs in CA1 SR interneurons voltage-clamped at +40 mV (Fig. 3E). Choline at the concentration tested would have fully activated 7 nAChRs and partially activated 34 nAChRs. The nAChR agonists ACh, nicotine, and cytisine, each at 0.1 mM, were capable of triggering EPSCs in CA1 SR interneurons (Fig. 3E). Measurements of the net charge carried by the EPSCs indicated that the magnitude of the responses elicited by ACh, nicotine, and cytisine was not statistically different from that of the response induced by DMPP (Fig. 3E). The order of agonist potency in inducing EPSCs was nicotine = cytisine = ACh = DMPP >> choline. A similar rank order of potency has been reported for agonist-induced activation of 34 nAChRs ectopically expressed in cells (Nelson et al. 2001; Quick et al. 1999; reviewed in Vizi and Lendvai 1999). Although studies carried out in primary hippocampal cultures, mouse brain synaptosomes, and ectopically expressing systems have reported that cytisine activates 42 nAChRs with poor efficacy (Alkondon and Albuquerque 1995; Lu et al. 1998; Nelson et al. 2001; Papke and Heinemann 1994), no information is available regarding the effectiveness of cytisine in activating 42* nAChRs in hippocampal slices of postnatal rats. In CA1 SR interneurons of rat hippocampal slices, ACh has been shown to activate slowly decaying currents that, being sensitive to blockade by DHE, are likely to be mediated by 42* nAChRs (Alkondon et al. 1999; Shao and Yakel 2000; Sudweeks and Yakel 2000). Here, evidence is provided that in CA1 SR interneurons of the rat hippocampal slices DMPP also activates DHE-sensitive whole cell currents and that cytisine does not fully activate these currents (Fig. 3, F and G). Thus, also in postnatal rat hippocampal neurons, cytisine acts as a poor agonist at 42* nAChRs.
In the absence of extracellular Mg2+, nicotinic agonist-triggered EPSCs increase the excitability of CA1 SR interneurons
To determine whether in conditions that decrease Mg2+-induced block of NMDA receptors phasic NMDA currents induced by nicotinic agonists can increase the excitability of CA1 SR interneurons, responses to DMPP or ACh were recorded from interneurons under cell-attached configuration using nominally Mg2+-free ACSF. MLA (10 nM) was included in the ACSF to prevent activation of somatodendritic 7* nAChRs by the agonists. The absence of extracellular Mg2+ mimics a scenario in which Mg2+ blockade of NMDA receptors is removed by a strong depolarizing stimulus. Most experiments consisted of three steps. First, DMPP was applied for 12 s to a region including and surrounding SR interneurons under cell-attached configuration and fast current transients that represented action potentials were recorded. Second, action potentials evoked by DMPP were recorded from the same neurons in the presence of the NMDA receptor antagonist APV (50 µM). Third, responses to DMPP were recorded after washing of APV. In some experiments, after the third step, the whole cell configuration was achieved and DMPP-evoked responses were recorded at –68 mV.
In the continuous presence of MLA (10 nM), application of DMPP (0.1 mM) to CA1 SR interneurons elicited action potentials and the frequency of events triggered by DMPP was significantly reduced by APV (Fig. 4A); the inhibitory effect of APV was reversed on washing of the slices with APV-free ACSF (Fig. 4A). On average, in nominally Mg2+-free ACSF, APV decreased the frequency of action potentials induced by DMPP or ACh by 60.3 ± 11.9% (mean ± SE; n = 7 neurons). In addition, DMPP-triggered NMDA EPSCs were recorded under whole cell configuration from neurons that under cell-attached configuration responded to the nicotinic agonist with APV-sensitive action potentials (Fig. 4A). These findings suggest that, under conditions that decrease Mg2+-induced block of NMDA receptors, glutamate released because of activation of nAChRs increase the excitability of the CA1 SR interneurons by enhancing the activity of their somatodendritic NMDA receptors.
|
In the absence of MLA, application of choline (10 mM) to the region surrounding and including CA1 SR interneurons also triggered bursts of action potentials (Fig. 4B). The frequency of choline-triggered action potentials recorded in the presence of APV was 97 ± 2.6% of that recorded in the absence of the NMDA receptor antagonist (n = 3 neurons; Fig. 4B). In addition, whole cell currents mediated by activation of somatodendritic 7* nAChRs were the predominant responses recorded under the whole cell configuration from interneurons that under the cell-attached configuration responded to choline with bursts of action potentials; only a few choline-triggered EPSCs could be recorded from these neurons (Fig. 4B). These results confirmed the notion that there is a direct relationship between the ability of the agonist to induce NMDA EPSCs and that of the agonist to evoke APV-sensitive action potentials. Thus choline-induced action potentials can be attributed to its well-known agonistic effect on somatodendritic 7* nAChRs in the SR interneurons (Alkondon et al. 1999; Frazier et al. 1998a; Ji and Dani 2000; Jones and Yakel 1997; McQuiston and Madison 1999).
In the presence of extracellular Mg2+, nicotinic agonists increase the excitability of CA1 SR interneurons by different mechanisms
To determine the contribution of different nAChR subtypes to regulation of excitability of CA1 interneurons under near physiological conditions, ACSF containing 1 mM Mg2+ was used to record choline- or DMPP-induced action potentials from interneurons in the cell-attached configuration. In 3 out of 4 interneurons continuously perfused with ACSF containing MLA (10 nM), DMPP induced no action potentials (data not shown). In one interneuron, which showed no spontaneous action potentials during a 3-min recording, DMPP triggered action potentials (Fig. 5Aa) that could not be blocked by APV (Fig. 5, Ab and c). When the whole cell configuration was achieved in all 4 neurons, DMPP induced both AMPA and NMDA EPSCs that were recorded prominently at –68 and +40 mV, respectively (Fig. 5, Ad and e). Considering that no nicotinic whole cell currents were detected in these neurons, it is likely that the action potentials triggered by DMPP were the result of activation of AMPA receptors by glutamate released from glutamatergic fibers/neurons synapsing onto the interneurons under study. These results support the notion that under physiological conditions nAChR-induced AMPA EPSCs alone can trigger action potential firing in the SR interneurons.
|
When the experiments were carried out in the absence of MLA, DMPP induced short-lasting bursts of action potentials in 6 out of 7 CA1 SR interneurons under cell-attached configuration (Fig. 5Ba). DMPP-triggered bursts of action potentials were not sensitive to block by APV (Fig. 5, Bb and c). Under the whole cell configuration, these neurons responded to DMPP with an 7* nAChR-mediated whole cell current at –68 mV and bursts of NMDA EPSCs at +40 mV (Fig. 5, Bd and e). Likewise, choline (10 mM) triggered bursts of APV-insensitive action potentials in CA1 interneurons under cell-attached configuration (n = 4 neurons). These findings indicate that activation of somatodendritic 7* nAChRs, although sufficiently strong to reach the threshold of action potential firing in the interneurons, is unable to cause the sustained depolarization required to relieve Mg2+ block of NMDA receptors.
CA1 SR interneurons that under cell-attached configuration responded to 0.1 mM DMPP with long-lasting bursts of action potentials sensitive to partial blockade by APV (Fig. 5, Ca and b) showed at –68 mV slowly decaying whole cell currents in response to the same concentration of DMPP (Fig. 5Cc). These CA1 SR interneurons belonged to the interneuron population that respond to nicotinic agonists with slowly decaying currents that are sensitive to block by DHE and are thus mediated by activation of somatodendritic 42* nAChRs. This population corresponded to approximately 28.5% of the CA1 SR interneurons (35 out of 123 neurons). Application of DMPP to these interneurons voltage-clamped at +40 mV triggered NMDA EPSCs (Fig. 5Cd). These results suggest that activation of somatodendritic 42* nAChRs causes sufficiently prolonged neuronal depolarization to relieve the Mg2+ block of NMDA receptors, thus enabling NMDA EPSCs to reach threshold for action potential firing.
Removal of extracellular Mg2+ reveals the presence of tonic NMDA currents that increase the excitability of CA1 SR interneurons
Comparison of whole cell currents recorded in the absence and in the presence of 1 mM extracellular Mg2+ indicated that the basal current fluctuation was increased by removal of Mg2+ (see Fig. 6A). Perfusion of the slices with CNQX (10 µM)-containing, nominally Mg2+-free ACSF did not alter the baseline current noise to any significant extent (data not shown). However, after a 2-min perfusion of the slices with APV-containing ACSF, the baseline current noise was significantly decreased (Fig. 6, A and B).
|
The magnitude of the tonic currents, as measured by the SD of the baseline current, was significantly larger in recordings obtained using nominally Mg2+-free ACSF than in recordings obtained using ACSF containing 1 mM Mg2+ (Fig. 6B). Further, APV significantly decreased the SD values of the baseline current recorded using the nominally Mg2+-free ACSF (Fig. 6B). These results suggest that in CA1 SR interneurons a tonic NMDA current is normally suppressed at the resting membrane potentials by physiological levels of Mg2+.
The influence of tonic NMDA currents on the excitability of CA1 SR interneurons was evaluated by measuring the frequency of spontaneous action potentials in cell-attached recordings in various experimental conditions (Fig. 6, C and D). All recordings were obtained using either nominally Mg2+-free or Mg2+-containing ACSF. The frequency of action potentials recorded from interneurons in the absence of Mg2+ was significantly higher than that recorded in the presence of Mg2+ (Fig. 6, C and D). Further, APV (50 µM) attenuated significantly the enhancement of frequency of action potentials that was caused by removal of extracellular Mg2+, although it could not bring the frequency of events back to control values (Fig. 6C).
To investigate whether nAChR activation would modify tonic NMDA currents, whole cell recordings were obtained from CA1 SR interneurons at +40 mV in Mg2+-containing ACSF. At this membrane potential, contamination of the recordings with slowly decaying nicotinic currents became minimal because of strong rectification. In addition, MLA was used to inhibit activation of 7* nAChRs, which could have produced some outward nicotinic currents. Neurons that exhibited high frequency of NMDA EPSCs in response to nicotinic agonists were not used because the phasic NMDA activation interferes with the analysis of tonic NMDA currents. Under these experimental conditions, DMPP (Fig. 7B) or ACh failed to induce tonic NMDA currents in neurons that showed some phasic events triggered by the agonists. The SD values for the baseline current at +40 mV did not increase significantly during exposure to the agonists (Fig. 7C). These results indicated failure of nAChR activation by exogenously applied agonists to enhance tonic NMDA currents in CA1 SR interneurons.
|
As shown in Fig. 7, A and C, exposure of the slices to APV decreased to a significant extent the SD of the baseline current recorded from CA1 SR interneurons voltage-clamped at +40 mV. In contrast, perfusion of the slices with ACSF containing TTX, mecamylamine, or MLA failed to affect the SD of the baseline current recorded from CA1 SR interneurons (Fig. 7C). Thus modifying quantal glutamate release by means of TTX-induced Na+-channel block, exogenously applied nicotinic agonists, or endogenous nAChR activity does not affect the tonic NMDA receptor activity, suggesting that the tonic NMDA receptor activity results from the actions of ambient levels of glutamate on extrasynaptic NMDA receptors (Sah et al. 1989).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
7*, 42*, and 34* nAChRs modulate synaptic inputs to CA1 SR interneurons
The presence of 7* nAChRs in interneurons of the CA1 field of the rat hippocampus has been well documented (Alkondon et al. 1997, 1999; Frazier et al. 1998a; Ji and Dani 2000; Jones and Yakel 1997; McQuiston and Madison 1999). In the SR of the CA1 field of the hippocampus, approximately 90% of the interneurons express 7* nAChRs (Alkondon and Albuquerque 2001); these receptors mediate fast nicotinic cholinergic synaptic transmission, fast-decaying nicotinic currents (referred to as type IA), and short bursts of action potentials (Alkondon et al. 1998, 2000; Frazier et al. 1998b). In addition, short bursts of GABAergic IPSCs can be recorded from various types of CA1 neurons that receive innervation from the 7* nAChRs-expressing interneurons (Alkondon and Albuquerque 2001; Ji and Dani 2000). 42* nAChRs are also present in CA1 interneurons of different strata, including SR interneurons (Alkondon et al. 1997, 1999; Jones and Yakel 1997; McQuiston and Madison 1999; Sudweeks and Yakel 2000). However, activation of 42* nAChRs triggers slowly decaying nicotinic currents as well as long bursts of action potentials (referred to as type II) in CA1 SR interneurons, and evokes long-bursts of GABAergic IPSCs that can be recorded from CA1 neurons that receive innervation from the 42* nAChR-expressing interneurons (Alkondon and Albuquerque 2001; Alkondon et al. 1999; McQuiston and Madison 1999). In the present study, evidence is provided that CA1 SR interneurons expressing 42* nAChRs correspond to approximately 28% of the CA1 SR interneuronal population. In addition, this study shows that cytisine, a nicotinic agonist known to act as a partial agonist on 42* nAChRs in various systems (Alkondon and Albuquerque 1995; Lu et al. 1998; Luetje and Patrick 1991; Nelson et al. 2001; Papke and Heinemann 1994), is a poor agonist on 42* nAChRs present in CA1 SR interneurons of young rats.
The findings that 1) TTX-sensitive EPSCs could be recorded from CA1 SR interneurons in response to nicotinic agonists; 2) MLA, an 7 nAChR antagonist, failed to inhibit EPSCs triggered by the nicotinic agonists ACh and DMPP; 3) choline, an 7 nAChR-preferring agonist, was the weakest agonist to induce the EPSCs; and 4) cytisine, a weak agonist at 42 nAChRs, was as efficacious as ACh and DMPP in inducing EPSCs in SR interneurons all support the previous demonstration that 34* nAChR activation in pyramidal neurons/fibers facilitates glutamate release onto the interneurons (Alkondon and Albuquerque 2002). This conclusion is further strengthened by the observation that mecamylamine at concentrations between 0.1 and 1 µM was very effective in inhibiting DMPP-induced NMDA EPSCs. We estimate that the IC50 of mecamylamine is between 0.1 and 0.3 µM, and a similar range of concentrations has been reported to inhibit specifically 34 nAChR with negligible effect on 7 or 42 nAChRs expressed in Xenopus oocytes (Papke et al. 2001).
In addition to increasing the phasic AMPA receptor activity (see also Alkondon and Albuquerque 2002), facilitation of glutamate release onto CA1 interneurons by 34* nAChR activation in glutamatergic neurons enhanced the phasic NMDA receptor activity in the interneurons. The increased quantal release of glutamate contributed to the recruitment of the phasic NMDA receptor activity, as evidenced by the positive correlation between the total amplitude and the net charge of each nicotinic agonist-induced EPSC. Considering that bursts of EPSCs recorded from the SR interneurons resulted from similar bursts of action potentials in the presynaptic glutamatergic neurons, it can be inferred that NMDA receptor activation on the interneurons was favored by the build up of glutamate concentrations at the glutamatergic synapses in response to bursts of action potentials generated at the glutamatergic neurons as a consequence of the nAChR activation. In fact, it has been reported that bursts of action potentials increase the reliability of synaptic transmission in different areas of the brain, including the hippocampus (Lisman 1997).
nAChRs, AMPA, and NMDA receptor activation in different neuronal compartments regulates the overall excitability of CA1 SR interneurons
Although the origin of the nAChR-containing glutamatergic neurons is not identified in this study, the following lines of evidence indicate that they might be CA1 pyramidal neurons. First, the presence of a slowly decaying nicotinic current sensitive to blockade by mecamylamine in CA1 pyramidal neurons was recently demonstrated (Ji et al. 2001). Second, CA1 pyramidal neurons are known to send feedback projections to CA1 SR interneurons (Grunze et al. 1996; Lacaille and Schwartzkroin 1988; Williams et al. 1994). Alternatively, SR giant cells (Gulyas et al. 1998; Kirson and Yaari 2000) could be the 34* nAChR-expressing neurons that provide recurrent glutamatergic inputs to the SR interneurons. This possibility is supported by the report that CA1 SR giant cells are glutamatergic and resemble pyramidal neurons (Gulyas et al. 1998). In a simple neuronal circuitry composed of an nAChR-expressing pyramidal neuron synapsing onto interneurons, nAChR activation increases the firing of the pyramidal cell that in turn releases glutamate, which then activates AMPA and NMDA receptors in SR interneurons (see Fig. 8). Considering that the AMPA receptor antagonist CNQX caused a larger inhibition of DMPP-induced EPSCs than one would have expected from the block of postsynaptic AMPA receptors in the interneurons, it can be concluded that nAChR-induced glutamate release involved the indirect participation of AMPA receptors.
|
Thus at least two case scenarios can be envisioned. In one, AMPA receptors located in presynaptic terminals of a pyramidal neuron that synapses onto the interneuron are activated by glutamate released on activation of the 34* nAChRs and thus boost the release of glutamate (see Fig. 8). The other scenario considers synapses between pyramidal neurons expressing both AMPA receptors and 34* nAChRs and interneurons. It is feasible that under basal conditions, the level of AMPA receptor activity in the pyramidal neuron is not sufficient to cause substantial firing and glutamate release. However, any degree of depolarization resulting from the 34 nAChR activation can add to the AMPA receptor–induced depolarization and cause the pyramidal neuron to fire more frequently, and therefore to release more glutamate onto the interneuron under study (Fig. 8). The two possibilities are supported by the facts that 1) AMPA receptors located in presynaptic terminals of hippocampal neurons are known to facilitate glutamate release (Barnes et al. 1994) and 2) slowly decaying currents that are likely to be mediated by 34* nAChRs have been recorded from CA1 pyramidal neurons in rat hippocampal slices (Ji et al. 2001).
Several lines of evidence provided herein indicate that nicotinic agonists regulate the excitability of the CA1 SR interneurons by distinct mechanisms. In 7* nAChR-expressing interneurons, neuronal excitability is regulated by nicotinic agonists primarily by an NMDA receptor-independent mechanism that involves the direct activation of somatodendritic 7* nAChRs and, possibly, the indirect activation of somatodendritic AMPA receptors by glutamate released on activation of 34* nAChRs in the pyramidal neurons. On the other hand, in 42* nAChR-expressing interneurons, neuronal excitability is increased by nicotinic agonists by an NMDA receptor–dependent mechanism that involves the concomitant activation of presynaptically located 34* nAChRs and somatodendritic 42* nAChRs. In these neurons, depolarization induced by the 42* nAChRs is sufficiently strong and prolonged to release Mg2+-induced block of the NMDA receptors.
Previous studies have shown that long-term potentiation can be easily induced in pyramidal neurons and in SR giant cells, but not in SR interneurons (Gulyás et al. 1998; McBain et al. 1999; McMahon and Kauer 1997). The results presented herein indicate that only a small population of interneurons can be excited by coincidental activation of somatodendritic nAChRs and NMDA receptors. Therefore the phasic NMDA receptor activity observed in all CA1 SR interneurons as a consequence of glutamate released by the activation of 34* nAChRs in pyramidal neurons may subserve other physiological functions that are regulated by nAChRs and dependent on Ca2+ influx through NMDA receptors, including neuronal survival (Hardingham et al. 2002).
Significance of tonic NMDA activity in the hippocampal CA1 region
Reduction of extracellular levels of Mg2+ in hippocampal slices has been used as an in vitro model of enhanced electrical noise arising from increased tonic NMDA receptor activation (Danysz et al. 1995; Frankiewicz and Parsons 1999; Sah et al. 1989). The use of nominally Mg2+-free ACSF revealed that the excitability of CA1 SR interneurons is modulated by tonic NMDA receptor activity. The finding that TTX- and nicotinic agonist–induced changes in quantal glutamate release failed to alter the basal current noise recorded from the interneurons is in agreement with the concept that tonic NMDA receptor activity results from the actions of ambient levels of glutamate on extrasynaptic NMDA receptors (Sah et al. 1989).
In CA1 pyramidal neurons, tonic NMDA receptor activation was reported to facilitate discharge of action potentials and enhance coupling between dendritic excitatory synaptic inputs and somatic action potential discharge (Sah et al. 1989). Because tonic NMDA receptor activation also enhanced firing of CA1 SR interneurons, modulation of this tonic glutamatergic activity can potentially alter the input–output properties of the hippocampus and contribute to both the hyperexcitability seen during epileptic discharges and the cell death observed in a number of neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. In this regard, it is noteworthy that increased phasic NMDA receptor activity decreases, whereas tonic NMDA receptor activity enhances apoptosis of hippocampal neurons by the CREB shutoff pathway (Hardingham et al. 2002). Thus selective enhancement of phasic NMDA receptor activity in CA1 SR interneurons attributed to activation of 34* nAChRs located on glutamatergic axons/fibers can contribute to the reported antiapoptotic properties of nicotinic agonists (Dajas-Bailador et al. 2000).
In conclusion, the present study reveals that the cholinergic and the glutamatergic systems interact to regulate the excitability of CA1 SR interneurons by mechanisms that involve the participation of nAChRs, NMDA, and AMPA receptors present in distinct neuronal compartments. A temporal and spatial integration of these signals can form the basis for the origin of hippocampal rhythms that can be triggered and/or modulated by nicotinic agonists and are essential for learning and memory (Bland and Colom 1988; Cobb et al. 1999).
|
DISCLOSURES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
1 According to the current status of the nomenclature for nAChRs and their subunits (Lukas et al. 1999), the asterisk is meant to indicate that the exact receptor subunit composition is not known. 
Address for reprint requests: E. X. Albuquerque, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: ealbuque{at}umaryland.edu).
|
REFERENCES |
|---|
