Properties of Neuronal Nicotinic Acetylcholine Receptors

The 1996 ASPET Otto Krayer Award Lecture

Properties of Neuronal Nicotinic Acetylcholine Receptors:Pharmacological Characterization and Modulation ofSynaptic Function1,2

EDSON X. ALBUQUERQUE, MANICKAVASAGOM ALKONDON, EDNA F. R. PEREIRA, NEWTON G. CASTRO,ANDRE SCHRATTENHOLZ, CATAO T. F. BARBOSA, RAFAEL BONFANTE-CABARCAS, YASCO ARACAVA,HOWARD M. EISENBERG and ALFRED MAELICKE

Department of Pharmacology and Experimental Therapeutics (E.X.A., M.A., R.B.C., C.T.F.B., E.F.R.P.) and Department of Neurosurgery(H.M.E.), University of Maryland School of Medicine, Baltimore, Maryland; Department of Clinical and Basic Pharmacology (Y.A., N.G.C.,E.X.A.), and Laboratory of Molecular Pharmacology, Institute of Biophysics Carlos Chagas Filho (E.X.A., Y.A., N.G.C., R.B.C., C.T.F.B.)Univ. Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944, Brazil; Department of Biochemistry and Pathobiochemistry (A.M., A.S.), Univ. MainzSch. Med., Mainz, D-5500, Germany

Accepted for publication December 20, 1996

Mr. Chairman, ladies and gentlemen, it is an honor toreceive from the American Society of Pharmacology and Ex-perimental Therapeutics the 1996 Otto Krayer Award spon-sored by Zeneca Pharmaceutics Co. I am especially delightedto receive this award not only because of the remarkablecontributions that Prof. Krayer gave to Pharmacology, par-ticularly his studies on the hypotensive alkaloids from vera-trum, but far more important, because of the superior moraland ethical principles he demonstrated by refusing to acceptthe position of Professor and Head of Pharmacology at Dus-seldorf University after Philip Ellinger had been removed bythe Nazi government. I quote, “More important perhaps forthe history of the time was Krayer’s unparalleled audacity in1933 when he wrote a letter to the all-powerful PrussianMinister of Education in which he explained why he feltunable to accept the chair of pharmacology at Dusseldorffrom which the Jewish incumbent, Philip Ellinger, had just

been removed: ‘. . .the primary reason for my reluctance isthat I feel the exclusion of Jewish scientists to be an injustice,the necessity of which I cannot understand, since it has beenjustified by reasons that lie outside the domain of science.This feeling of injustice is an ethical phenomenon. It is innateto the structure of my personality, and not something im-posed from the outside. Under these circumstances, assum-ing the position would make it difficult to take up my dutiesas teacher with joy and a sense of dedication, without whichI cannot teach properly. . . The work to which I have hereto-fore dedicated all my strength, means so much to me that Icould not compromise it with the least bit of dishonesty. Itherefore prefer to forego this appointment, rather than hav-ing to betray my convictions; or that by remaining silent Iwould encourage an opinion about me that does not corre-spond with the facts.’ Prof. Krayer was summarily dismissedand informed that all German Universities henceforth were‘off-limits’ for him. In 1937 he joined the Faculty of HarvardMedical School as Associate Professor of Pharmacology andtwo years later succeeded Reid Hunt (1870–1948) as Head ofthe Department.” (Witkop, 1995).This lecture, delivered in March 1996 during the Annual

Meeting of the American Society of Pharmacology and Ex-perimental Therapeutics, brings together many publishedand some unpublished findings from our laboratories on the

Received for publication December 19, 1996.1 Otto Krayer Award Lecture in Pharmacology and Experimental Thera-

peutics presented by the recipient, Dr. Edson X. Albuquerque.2 This work was supported in part by National Institutes of Health grant

NS25296, Environmental Health Service grant ES05730, U.S. Army MedicalResearch and Development Command Contracts DAMD17-95-C-5063, FINEP,CNPq and PRONEX (Brazil), and the University of Maryland at Baltimore/Federal University of Rio de Janeiro Molecular Pharmacology Training Pro-gram.

ABBREVIATIONS: nAChRs, nicotinic acetylcholine receptors; CNS, central nervous system; a-BGT, a-bungarotoxin; MLA, methyllycaconitine;a-CTx-ImI, a-conotoxin-ImI; DHbE, dihydro-b-erythroidine; DMPP, dimethylphenylpiperazinium; a-CTxs, a-conotoxins; AChE, acetylcholinest-erase; VR, reversal potential; ATP-R internal solution, adenosine trisphosphate-regenerating internal solution; NMDA, N-methyl-D-aspartate;[Ca11]o, extracellular concentrations of Ca

11; GABAA receptor, type A g-aminobutyric acid receptor; M10 cells, mammalian fibroblasts that stablyexpress the a4b2 nAChR; 5-HT, 5-hydroxytryptamine; APV, D,L-2-amino-5-phosphonovaleric acid; LTP, long-term potentiation; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; TTX, tetrodotoxin; ACSF, artificial cerebrospinal fluid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.

0022-3565/97/2803-1117$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 3Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.JPET 280:1117–1136, 1997

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properties of neuronal nAChRs in the mammalian CNS thathave led the way to a better understanding of the nAChRfunction in the brain.Neuronal nAChR subtypes in the CNS are extremely di-

verse in their kinetics of activation and inactivation and intheir sensitivity to nicotinic agonists and antagonists (Sar-gent, 1993; Lindstrom, 1995; Role and Berg, 1996; Albuquer-que et al., 1995a,b; Albuquerque et al., 1996). This diversity,which may provide the basis by which several physiologicalactions can be triggered by a single neurotransmitter, is aconsequence of the various subunit compositions of thesereceptors that can be heterooligomers formed by combina-tions of agonist-binding a subunits (a2–a9) with structural bsubunits (b2–b4), or homoligomers formed by the a7, a8 ora9 subunit. Numerous studies in expression systems such asoocytes, fibroblasts, COS and HEK cells have dealt with thecharacterization of the possible functional receptors formedsolely by a subunits or by combinations of a and b subunits(Bertrand et al., 1992; Seguela et al., 1993; Elgoyhen et al.,1994; Gerzanich et al., 1994; Peng et al., 1994; Briggs et al.,1995). However, it was not until recently that clues wereprovided about the possible physiological roles of the variousneuronal nAChR subtypes in the brain.One of the major problems in addressing nAChR function

in the brain has been the lack of specific agonists and antag-onists for each of the receptor subtypes (for a review, seeAlbuquerque et al., 1995a,b). This problem was aggravatedby the fast kinetics of inactivation of some neuronal nAChRs,particularly those composed of the a7 subunit. The field hasnow advanced immensely, and many of these problems havebeen overcome. In this lecture, studies from our laboratorieswill be presented chronologically from the initial character-ization of the diverse subtypes of native nAChRs in the CNSto the identification of the physiological functions of some ofthese receptor subtypes, including the description of the re-ceptor distribution on the surface of neurons.

Characterization of the nAChR SubtypesUnderlying the Responses of Hippocampal

Neurons to Nicotinic Agonists andAntagonists

It is generally acknowledged that the hippocampus, anarea of the CNS that is involved in processing cognitivefunctions (Petit, 1988), is very sensitive to cholinergic mod-ulation, and that the density of neuronal nAChRs in thehippocampus is severely diminished in diseases character-ized by learning and memory impairment (e.g., Alzheimer’sdisease) (Maelicke and Albuquerque, 1996). Actually, it isfair to say, “The hippocampus’s main input is in providing akeen memory of context, vital for emotional meaning; it is thehippocampus that recognizes the differing significance of,say, a bear in the zoo versus one in your backyard” (Goleman,1995). Thus, the developing hippocampus has become thefocus of our interest with regard to characterization of thenAChR function; and electrophysiological techniques, partic-ularly the patch-clamp technique, have been remarkably im-portant for the studies of the functional and pharmacologicalproperties of nAChRs expressed in single neurons.Hippocampal neurons in culture respond to nicotinic ago-

nists with one of three types of nicotinic whole-cell currents,named type IA, type II and type III, which are distinguished

from one another on the basis of their kinetic and pharma-cological properties (fig. 1; Alkondon and Albuquerque,1993). Type IA currents, by far the predominant response ofhippocampal neurons to nicotinic agonists, are fast-desensi-tizing currents that show a rundown that is associated withintracellular high-energy phosphate compounds and an in-tracellular Mg11-dependent inward rectification (fig. 1;Alkondon and Albuquerque, 1993; Alkondon et al., 1994;Castro and Albuquerque, 1995). The fast kinetics of inacti-vation and the short-lived open time of the nAChR channelsthat subserve type IA currents account for the unique kineticproperties of these currents (Castro and Albuquerque, 1993).Further, type IA currents have a high sensitivity to blockadeby a-BGT, MLA, a-CTx-ImI and a-cobratoxin (fig. 1) (Alkon-don et al., 1992; Alkondon and Albuquerque, 1990, 1991,1993; Pereira et al., 1996).In contrast to type IA currents, type II and III currents,

which desensitize very slowly, can be recorded from a smallpopulation of the hippocampal neurons (Alkondon and Albu-querque, 1993, 1995). Also in contrast to type IA currents,type II currents show an inward rectification that is indepen-dent of intracellular Mg11 and do not run down (fig. 1)(Alkondon et al., 1994). Approximately 10% of the hippocam-pal neurons in culture respond to nicotinic agonists with typeII currents, whereas no more than 2% of the neurons inculture respond to the agonists with type III currents. Type IIand III currents are differentiated from one another on thebasis of their sensitivity to nicotinic antagonists. Activationof type II currents is inhibited by DHbE (10 nM), and acti-vation of type III currents is inhibited by mecamylamine(1 mM) (fig. 1).A comparison of the kinetic and pharmacological proper-

ties of the nicotinic currents evoked in hippocampal neuronsto those of currents elicited in oocytes ectopically expressingdistinct subtypes of functional nAChRs led to the suggestionsthat an a7-bearing nAChR subserves type IA currents, ana4b2 nAChR subserves type II currents and an a3b4 nAChRsubserves type III currents. These suggestions were sup-ported not only by the finding of mRNAs coding for a7, a4and b2 subunits in hippocampal neurons, but also by theproportion of cultured hippocampal neurons that bind[125I]a-BGT (a probe to label a-BGT-sensitive neuronalnAChRs) or [3H]nicotine (a probe that labels the high-affin-ity, presumably a4b2 neuronal nAChRs) (Alkondon et al.,1994; Barrantes et al., 1995).Analysis of the stoichiometry of some neuronal nAChRs

have indicated that, similarly to muscle nAChRs, those re-ceptors are pentameric and composed of two a subunits andthree b subunits (Cooper et al., 1991). However, there isevidence that some neuronal nAChRs may be composed of asmany as three different subunits (Vernallis et al., 1993), andthat some subunits (a7, a8 or a9) are capable of forminghomomeric nAChRs that are functional when heterologouslyexpressed in systems such as oocytes or fibroblasts [for areview see Lindstrom (1995)]. Thus, one cannot conclude byanalogy that all neuronal nAChRs will necessarily have twoagonist-binding a subunits and three structural b subunits.It is also difficult to conclude on a comparative basis thatnative nAChRs have exactly the same subunit compositionand arrangement as those described for heterologously ex-pressed nAChRs. For instance, it is still questionablewhether native a7-containing receptors are homomeric.

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Analysis of the efficacy and potency of various agonists re-vealed that the EC50 values for ACh, nicotine and cytisine inactivating type IA currents are approximately 130, 27 and 50

mM, respectively (fig. 2) (Alkondon and Albuquerque, 1993,1995), whereas the EC50 values for these compounds in acti-vating nicotinic currents in Xenopus oocytes expressing the

Fig. 1. Pharmacological and kinetic properties of nicotinic currents recorded from hippocampal neurons. Top row illustrates the typical family ofwhole-cell currents (type IA, type II and type III) evoked by application of ACh to hippocampal neurons and their sensitivity to blockade by nicotinicantagonists. Short pulses (1–2 sec) of ACh (3 mM) were applied via a U-tube to hippocampal neurons at the time indicated by downward arrows.Antagonists were applied via bath superfusion and as an admixture with ACh. Holding potential 5 256 mV. Middle row depicts the rundown withtime of peak amplitude of type IA currents and its prevention by the use of an ATP-regenerating internal solution [see Alkondon et al. (1994) fordetailed solution compositions]. Graph on the right illustrates that, in contrast to type IA currents, type II currents do not run down. Bottom rowdisplays the current-voltage plots of type IA (left) and type II currents (right). Type IA currents only showed inward rectification when MgCl2 wasadded to the internal recording solution, whereas type II currents showed inward rectification even in the absence of addedMg11 in the pipette solution.

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a7 homomers are about 112, 7.8 and 18 mM, respectively(Gerzanich et al., 1994). According to these results, nicotineand cytisine are at least 3-fold less potent in evoking type IA

current than in evoking nicotinic currents in Xenopus oocytesexpressing a7 homomers. In addition, DMPP acts as a fullagonist in eliciting type IA currents in rat hippocampal neu-rons (Alkondon and Albuquerque, 1993, 1995), whereas itacts as a very weak partial agonist in evoking nicotinic cur-rents through chick a7 nAChR homomers expressed in Xe-nopus oocytes (Gerzanich et al., 1994). Species-specific differ-ences in the sequence of these subunits could account forthese discrepancies, particularly because DMPP acts as a fullagonist in human neuroblastoma cells heterologously ex-pressing homomers of human nAChR a7 subunits (Peng etal., 1994). The discrepancies between the apparent potency ofan agonist in activating native a7 nAChRs and a7 nAChRsubunits expressed in Xenopus oocytes could also be ac-counted for by the fact that posttranslational modifications ofthe a7 subunits in oocytes may differ from those that occur inmammalian systems, resulting in modifications of some ofthe properties of the receptors heterologously expressed inoocytes (Siviloti et al., 1995). In fact, it has been reported thatprotein processing in mammalian systems can be differentfrom protein processing in the oocytes (Shi et al., 1994).Despite these explanations for the differences between thepharmacological properties of a-BGT-sensitive hippocampalnAChRs and those of homomers of a7 subunits heterolo-gously expressed in Xenopus oocytes, it is still possible thatthe native a-BGT-sensitive nAChR in hippocampal neuronsis a heteromeric receptor that bears the a7 subunit.The recent introduction of a-CTx-ImI as a competitive an-

tagonist selective for a-BGT-sensitive, a7-bearing nAChRs inhippocampal neurons may represent a step forward towardthe characterization of the structure of these native receptors(Pereira et al., 1996). a-CTxs, small peptides purified fromthe venom of Conus snails, are so named because they arepotent inhibitors of the activation of muscle nAChRs (Myerset al., 1991). Some of the a-CTxs, particularly a-CTx-GI anda-CTx-MI, were very useful for the dissection of the molecu-lar determinants of cholinergic binding sites on musclenAChRs (Groebe et al., 1995). Thus, a-CTx-ImI could be auseful tool for the analysis of the components of the bindingsites for competitive ACh antagonists on a7-bearing nativenAChRs, and could unveil the characteristics of the structureand subunit composition of the receptor.Studies of the efficacy and potency of various agonists in

activating different subtypes of neuronal nAChRs have led tothe fundamental discovery that choline, the metabolic prod-uct of ACh degradation in vivo, acts as an agonist as effica-cious as ACh at the a7 nAChRs in hippocampal neurons.Application of choline (10 mM) to hippocampal neurons thatrespond to ACh (3 mM) with type IA currents results inactivation of currents with the same characteristics as andamplitudes similar to those of the ACh-evoked currents (fig.2). The EC50 for choline in eliciting type IA currents inhippocampal neurons is about 1 mM (fig. 2). In contrast,choline (up to 10 mM) evokes no response in neurons thatrespond to ACh with type II currents, which indicates thatcholine does not activate the a4b2 nAChRs that subservethese currents (fig. 2).Some hippocampal neurons respond to ACh with a current

that is referred to as type IB and has a fast and a slowcomponent (fig. 3) (Alkondon and Albuquerque, 1993; Alkon-don et al., 1994). The fast component of type IB currents hasthe same pharmacological and kinetic properties as type IA

Fig. 2. Apparent potency of different agonists in eliciting type IA andtype II currents in hippocampal neurons. Top graph portrays the con-centration dependency of the activation by different nicotinic agonistsof type IA currents in hippocampal neurons. All responses are normal-ized to the response of the neurons to ACh (3 mM), which is taken as100%. Bottom graph illustrates the selectivity of nicotinic agonists inactivating type IA and type II currents. The extent of activation of typeIA currents at agonist concentrations which were effective in eliciting 0,10, 30, 50, 70 and 90% of the maximal amplitude in type II currents isshown. Same color code applies to both graphs.


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currents (fig. 3), which indicates that it is subserved by a7nAChRs. On the other hand, the properties of the slow com-ponent of type IB currents are the same as those of type IIcurrents (fig. 3), which indicates that it is subserved by a4b2nAChRs. In such neurons expressing both a7 and a4b2nAChRs, choline only activates the fast-decaying current,i.e., the current subserved by a7 nAChRs (fig. 3).At nAChR subtypes other than the a7 nAChRs, choline

acts as an extremely weak nicotinic agonist (Mandelzys et al.,1995). Thus, the concept that ACh hydrolysis is the means bywhich ACh activity ends in vivo appears to be true for thecholinergic functions mediated by most nAChR subtypes, butnot for those mediated by a7 nAChRs. Considering that cho-line uptake into the presynaptic terminal is a slow process,the concentration of choline in the synaptic cleft during syn-

aptic activity could be sufficient to lead to the activation of a7nAChRs. It is possible that cholinergic functions mediated bya7 nAChRs are limited or terminated by the agonist-inducedinactivation of these receptors. These findings altogetherbring novel concepts toward the development of therapeuticcompounds to treat diseases in which the a7 nAChR activityis reduced. It seems logical that the use of anti-AChE or offull nicotinic agonists would not be a great help in suchpathological conditions, unless a brief activation of the a7nAChRs preceding receptor desensitization would be enoughto trigger a cascade of long-lasting effects.

Functional Characteristics of the a7-BearingnAChR Channels Expressed in the

Hippocampus: Ca11 Permeability andModulation of Receptor Function by

Divalent CationsImportant clues about the possible physiological roles of

the a-BGT-sensitive, a7-containing nAChRs in the CNS wereobtained on the basis of the studies showing that these re-ceptors, similar to the homomeric a7 nAChRs expressed inXenopus oocytes, show a unique high permeability to Ca11

(Bertrand et al., 1993; Sands et al., 1993; Seguela et al., 1993;Castro and Albuquerque, 1995). The ion selectivity of thenative a-BGT-sensitive nAChRs in hippocampal neurons wasdetermined on the basis of the analysis of the reversal poten-tial (VR) of ACh-induced type IA currents under various ionicconditions (fig. 4). Using physiological salt solutions withdifferent ion activities (table 1) and a Goldman-Hodgkin-Katz equation (equation 1) for VR shifts in the presence ofCa11, permeability ratios were calculated.

PCa

PCs5

Cso2 2 Cso1eDVR F/RT

4 Cao1 eDVR F/RT ~11 eVR1 F/RT!21 2 4 Cao2 ~11 eVR2 F/RT!21 ~1!

The VR values of ACh-evoked type IA currents were deter-mined by use of physiological salt solutions with various ionic

Fig. 3. Characterization of neurons that express both a7 and a4b2nAChRs. (top row) The fast component of ACh (3 mM)-evoked type IBcurrent is blocked by the a7 nAChR antagonist MLA (1 nM), whereasthe slow component is abolished by the a4b2 nAChR antagonist DHbE(100 nM). (bottom row) In another neuron that responds to ACh withtype IB currents, choline evokes only the type IA current, which isabolished by MLA.

Fig. 4. Comparison of the Ca11 per-meability of a7 nAChRs to that ofNMDA receptors in hippocampal neu-rons. (A) The reversal potential of ACh-evoked type IA currents becomesmore positive when the extracellularCa11 concentration is increased from1 to 10 mM. (B) The reversal potentialof NMDA-evoked currents is also dis-placed to a more positive value uponincreasing the extracellular Ca11 con-centration from 1 to 10 mM. The ATP-regenerating intracellular solution andthe Cs1-based external solution wereused in these experiments (see table 1for solution composition).


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compositions, and the ATP-R internal solution was used inthe recording pipette to prevent to a large extent the run-down of type IA currents. With this internal solution and thestandard external solution, the VR of ACh-evoked type IAcurrents was 3.9 6 0.3 mV (Castro and Albuquerque, 1995).The Ca11-dependent changes in VR were tested with Cs

1 asthe main cation on both sides of the membrane, and withphysiologically relevant Ca11 concentrations. With Cs1-based external solution containing Ca11 (1 mM), the VR ofACh-evoked type IA currents was 23.0 6 0.4 mV, and the VRNMDA-evoked currents was 22.7 6 0.3 mV (fig. 4). When theextracellular concentration of Ca11 was increased to 10 mM,the VR of the ACh-evoked currents was shifted by 5.6 6 0.4mV, and the VR of the NMDA current was shifted by 8.36 0.4mV (fig. 4). On the basis of the VR shifts, one can concludethat the a-BGT-sensitive, neuronal nAChR channel is highlypermeable to Ca11, although less than the NMDA receptor.Applying the shifts in VR to the GHK equation shown

above, and assuming that only Cs1, Na1, and Ca11 contrib-uted to the ACh- or NMDA-evoked currents, the permeabilityratios were estimated for the a-BGT-sensitive nAChRs andthe NMDA receptors. Substitution in equation 1 of the pairsof VR values obtained from experiments using the Cs1-basedexternal solution containing either 1 mM (VR1) or 10 mM(VR2) Ca

11 yielded an average PCa/PCs of 6.1 6 0.5 for theACh channel and of 10.3 6 0.7 for the NMDA channel. Thefraction of current carried by Ca11 can be estimated from thePCa/PCs using equation 2 (Spruston et al., 1995).

Pf 5 S1 1@M1#

@Ca21]oF1 2 exp ~2 FV/RT!

4 PCa/PCsGD21

(2)

In this equation, which assumes that the permeability to allmonovalent cations is the same and that the GHK equationcan describe the relationship between permeability and cur-rent, [M1] is the total activity of monovalent cations on eachside of the membrane and [Ca11]o is the extracellular activ-ity of Ca11. In the experiments designed to determine PCa/PCs in the presence of 1 mM Ca11, the extracellular Ca11

activity was 0.27, and the total activity of monovalent cationswas approximately 105. At room temperature, F/RT wastaken to be 0.04 mV21. Substituting these values and thepermeability values in equation 2, we estimate that close tothe resting potential of the hippocampal neurons (;250 mV)and in the presence of 1 mM extracellular Ca11, approxi-mately 5.6% of the ACh-evoked type IA current is carried byCa11, whereas approximately 9% of NMDA-evoked currentis carried by Ca11. Thus, theoretically the Ca11 entry intohippocampal neurons through a-BGT-sensitive nAChRs isequivalent to approximately 60% of that through NMDAreceptors. However, considering that the mean open time andthe kinetics of inactivation of the NMDA receptor channelare much slower than those of the a7 nAChRs in hippocam-

pal neurons (Castro and Albuquerque, 1993; Nelson and Al-buquerque, 1994), the Ca11 influx through the NMDA re-ceptor should be longer lasting than that through the a7nAChR. Therefore, it is likely that NMDA-type glutamatereceptors and native neuronal nAChRs made up of the a7subunits are involved in different Ca11 signaling pathways(Teyler et al., 1994).The a7 nAChRs in hippocampal neurons, in addition to

being permeable to Ca11, are also sensitive to changes in the[Ca11]o (Bonfante-Cabarcas et al., 1996). Extracellular Ca

11

modulates the affinity of the a7 nAChRs for ACh, the coop-erativity between ACh-binding sites, as well as the inwardrectification, the decay phase, and rundown of a7-nAChRs-mediated type IA currents (fig. 5). Upon increasing the[Ca11]o from 10 mM to 1 mM, the apparent affinity of the a7nAChRs for ACh increases and the cooperativity betweenACh-binding sites decreases. In the presence of 10 mM[Ca11]o, the values of EC50 and Hill coefficient for ACh ineliciting type IA currents are 289 6 51 mM and 2.7 6 0.2,respectively, whereas in the presence of 1 mM [Ca11]o thesevalues are 206 6 43 mM and 1.2 6 0.1, respectively. Furtherincrease of the [Ca11]o to 10 mM decreases the apparentpotency of ACh in evoking type IA currents and abolishes thecooperativity between the ACh-binding sites on the a7nAChRs. In the presence of 10 mM [Ca11]o, the EC50 and theHill coefficient for ACh in eliciting type IA currents are 262 662 mM and 1.00 6 0.1, respectively. Several lines of evidenceindicate that the effects of Ca11 on the interaction of AChwith the a7 nAChRs in the hippocampus are mediated by theinteractions of Ca11 with specific sites on the receptor ratherthan through nonspecific Ca11 sites on the membrane orthrough changes of surface potential (Bonfante-Cabarcas etal., 1996). Not only did the effects of Ca11 on the receptorfunction follow sigmoid functions, which is expected for ef-fects mediated by specific binding sites, but also calculatedsurface potential varied by less than 10% for different valuesof surface charge density. It is likely that binding of Ca11 tothe a7 nAChR in the hippocampus controls the concertedtransformation of the subunits to yield various open-channelstates (Bonfante-Cabarcas et al., 1996). This concept is inagreement with the multiple channel conductance states re-ported for the a7 nAChRs in hippocampal neurons (Castroand Albuquerque, 1993).The decay phase of type IA currents evoked by saturating

concentrations of ACh ($1 mM) is accounted for by the de-sensitization of the a7 nAChRs (Castro and Albuquerque,1993) and is accelerated by increasing the [Ca11]o from2mM to 10 mM (fig. 5). In the presence of 2 mM [Ca11]o, ACh(1 mM)-evoked type IA currents have a decay-time constantof about 20 msec, whereas in the presence of 10 mM [Ca11]o,the currents have a decay-time constant of about 10 msec(Castro and Albuquerque, 1995; Bonfante-Cabarcas et al.,

TABLE 1Ionic composition of the test solutions (in mM)

Na1 K1 Cs1 NMG1 Ca11 Mg11 Cl2 CH3SO32 F2

ExternalCs1-based, 1 mM Ca11 150 (105)a 35 1 (0.27) 83 100Cs1-based, 10 mM Ca11 150 (104) 20 10 (2.74) 86 100

InternalATP-R 159 (105) 5 70 60

a Numbers in parentheses represent the activity of an ion in the physiological salt solution.


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1996). Thus, extracellular Ca11 plays an important role inthe rate of desensitization of the a-BGT-sensitive, a7nAChRs. Changes in [Ca11]o also affected the inward recti-fication of type IA currents, which depends on the intracel-lular concentrations of Mg11. When recording type IA cur-rents using a F2-containing internal solution, no inwardrectification is observed. However, when a nominally Mg11-free, malate-based internal solution is used and the extracel-lular solution contains 2 mM Ca11, the rectification of typeIA currents is confined to a short range of membrane poten-tials (0–30 mV). On raising the intracellular concentration ofMg11 to 10 mM, the inward rectification persists up to50 mV; and, if concomitantly the [Ca11]o is lowered to 0.3mM or less, the rectification persists up to 70 mV (fig. 5).Thus, the inward rectification of type IA currents, which is

maximal in the presence of low extracellular Ca11 concen-trations and added intracellular Mg11, can be reversed whenthe extracellular Ca11 concentration is increased to levelsconsidered to be within the physiological range, which indi-cates that depending on the ongoing synaptic activity andlevels of Ca11 surrounding the a7 nAChR, the receptor ac-tivity at positive potentials can range from being negligible tobeing very high.

Modulation of the a7-nAChR Activity byAllosteric Ligands

The activity of many ligand-gated ion channels is subject tomodulation by ligands other than the natural agonist. Twotypical examples are noted: positive modulation of the

Fig. 5. Sensitivity of a7 nAChRs in hippocampal neurons to extracellular Ca11. (left, top graph) Rundown of type IA currents in the presence ofdifferent extracellular Ca11 concentrations. Holding potential, 250 mV. (right, top graph) Concentration-response relationship for ACh in evokingtype IA currents in the presence of various concentrations of extracellular Ca11. Holding potential, 250 mV. (bottom, left graph) Current-voltagerelationship for ACh (3 mM)-evoked type IA currents in the presence of different concentrations of extracellular Ca11. A malate-based internalsolution was used in these experiments [see Bonfante-Cabarcas et al. (1996) for detailed solution composition]. (bottom, right graph) Comparisonof the decay phase of ACh (3 mM)-evoked type IA currents in the presence of 1 or 10 mM extracellular Ca11. A 500-msec pulse of ACh was appliedto the neurons at the downward arrow. Various traces of currents recorded under each condition are aligned and superimposed. The amplitudeof all traces is normalized for comparison of the decay phase of the currents. The ATP-regenerating solution was used in this series of experiments.Holding potential, 256 mV.


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NMDA receptor activity by glycine (Johnson and Ascher,1992; Scatton, 1993), and of the GABAA receptor activity bybenzodiazepines and steroids (McDonald and Twyman,1992). There is increasing knowledge of allosteric ligandsthat control the activation of ligand-gated receptors such asthe GABAA receptors and the nAChRs, and very recently ourstudies have provided evidence for the existence of a site onthe nAChRs through which the receptor channel activity canbe potentiated by ligands referred to as noncompetitive ago-nists (fig. 6).In 1985, studies from this laboratory demonstrated that

some anti-AChE, particularly the carbamate physostigmine,activate the muscle nAChR in frog single muscle fibers andthat this effect was unrelated to the blockade of AChE (Shawet al., 1985; Albuquerque et al., 1988). At the time, a funda-mental concept emerged supporting the notion that, in addi-tion to blocking AChE, anti-AChE can also directly modifynAChR function, either by acting as open-channel blockers orby potentiating the nAChR activity. Subsequently, biochem-ical studies showed that physostigmine can also activate theTorpedo nAChR (Kuhlmann et al., 1991; Okonjo et al., 1991).A major discovery came with the demonstration that thenicotinic agonist action of physostigmine is not inhibited bycompetitive nicotinic antagonists, being sensitive only to in-hibition by the nAChR-specific monoclonal antibody FK1(Okonjo et al., 1991). These findings indicated that physostig-mine activates the nAChR channel by binding to a site dis-tinct from that for ACh and other classical nicotinic ligands.Photoaffinity labeling of the Torpedo nAChR with [3H]phy-sostigmine revealed that physostigmine binds to a region onthe nAChR a subunits that includes and/or surrounds theamino acid Lys-125. Given that the epitope for the antibodyFK1 is located on the amino acid sequence 118 to 142 on thenAChR a subunit (which is close to, but distinct from, thenAChR region to which ACh binds), and that this antibodyantagonizes the agonist action of physostigmine without af-fecting that of ACh, it is most likely that the ability ofphysostigmine to activate the muscle-type nAChR is medi-ated by its binding to the region including and surroundingthe amino acid Lys-125 on the nAChR a subunit.By electrophysiological techniques, we have been able to

demonstrate that the novel agonist effect of physostigmine isnot confined to the muscle nAChR. Physostigmine was shownto evoke single-channel currents when applied to outside-outpatches obtained from, 1) hippocampal neurons (which ex-press predominantly the fast-desensitizing, a7-bearing neu-ronal nAChRs), 2) mammalian fibroblasts (M10 cells) thatstably express the a4b2 nAChR upon induction with dexa-methasone, and 3) PC12 pheochromocytoma cells (which ex-press at least three subtypes of neuronal nAChRs) (figs. 7and 8) (Pereira et al., 1993a, 1994; Storch et al., 1995). Thesecurrents were characterized as nicotinic because, althoughbeing insensitive to blockade by competitive ACh antago-nists, they were sensitive to blockade by FK1 (figs. 7 and 8).Supporting the concept that physostigmine-evoked single-channel currents were nicotinic currents, physostigmine wasshown to be unable to evoke single-channel currents in out-side-out patches from M10 cells in which the nAChR expres-sion was not induced by dexamethasone (Pereira et al., 1994).The findings that physostigmine activates a variety ofnAChR subtypes and the region bearing the physostigmine-binding site, i.e., the region including and/or surrounding the

amino acid Lys-125 is well conserved among all the nAChR asubunits sequenced to date (Pereira et al., 1993a,b) suggestthat this novel binding site may be critical for the nAChRfunction in vivo. In addition to physostigmine, the anti-AChEgalanthamine, the muscle relaxant benzoquinonium and theopioid codeine, all of which are structurally related to phy-sostigmine, were found to activate the nAChR channel via

Fig. 6. Schematic representation of the GABAA receptor and the nAChRshowing the sites through which receptor activity can be modulated.Barb, barbiturates; PTX, picrotoxin; BDZ, benzodiazepines; NCA, non-competitive agonists (e.g., galanthamine); LA, local anesthetics; Ara,arachidonic acid.


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the physostigmine-binding site (Pereira et al., 1993a,b, 1994;Storch et al., 1995).To investigate the relevance of this physostigmine-binding

site for the nAChR function, physostigmine and structurallyrelated compounds were tested for their abilities to evokenicotinic whole-cell currents and to modulate the nAChRactivity induced by classical nicotinic agonists (Pereira etal., 1993a,b, 1994; Storch et al., 1995). Physostigmine and its1-methyl derivative, galanthamine and 1-methylgalan-

thamine, benzoquinonium and codeine were unable to evokenicotinic macroscopic currents (fig. 9). Instead, by binding tothe newly identified site on the nAChRs, physostigmine-likecompounds were shown to modulate ACh-induced nAChRactivity in different preparations (figs. 9 and 10). Galan-thamine and 1-methylgalanthamine increased the peak am-plitude of nicotinic whole-cell currents evoked by applicationof nonsaturating concentrations of ACh (or other classicalnicotinic agonists) to PC12 cells and cultured hippocampal

Fig. 7. Activation by physostigmine of nicotinic single-channel currents in outside-out patches excised from hippocampal neurons is inhibited byFK1. (left traces) Sample recordings of single-channel currents evoked by application of the classical nicotinic agonist (1)-anatoxin-a (AnTX) orby application of physostigmine (PHY) to outside-out patches excised from hippocampal neurons in culture. The agonist effect of PHY, but notthat of AnTX, is inhibited by FK1. (right graph) Quantification of the effect of FK1 on AnTX- and PHY-induced nAChR activity in hippocampalneurons. The frequency of channel activity evoked by the agonists in the absence of FK1 was taken as 100% and used to normalize the frequencyof channel activity recorded in the presence of FK1.

Fig. 8. Activation by AnTX, physostigmine and galanthamine of nicotinic single-channel currents in outside-out patches excised from M10 cellsexpressing a4b2 nAChRs. (top traces) Sample recordings of single-channel currents evoked by application of the classical nicotinic agonist(1)-anatoxin-a (AnTX), or by application of physostigmine (PHY) or galanthamine (GAL) to outside-out patches excised from fibroblasts stablyexpressing a4b2 nAChRs (M10 cells). Receptor expression in the M10 cells was induced by 3 to 5-day exposure of the cells to dexamethasone(1 mM). The agonist effect of GAL and PHY (not shown), but not that of AnTX, is inhibited by FK1. In contrast, the agonist effect of AnTX, but notthat of GAL or PHY (not shown), was sensitive to blockade by DHbE (30 nM), a competitive nicotinic antagonist specific for the a4b2 nAChRs.(bottom graph) Quantification of the effect of FK1 and DHbE on AnTX- and GAL-induced nAChR activity in hippocampal neurons. The frequencyof channel activity evoked by the agonists in the absence of antagonists was taken as 100% and used to normalize the frequency of channelactivity recorded in the presence of the antagonists.


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neurons, an effect that could be blocked by FK1 (figs. 9 and10). Also, by acting via the physostigmine-binding site, ga-lanthamine or its 1-methyl derivative was capable of pre-venting the a7-bearing nAChRs in hippocampal neurons andthe neuronal nAChRs expressed in PC12 cells from undergo-ing desensitization (figs. 9 and 10) (Schrattenholz et al.,1996). In this regard, the effect of galanthamine and relatedcompounds on the nAChRs resembles that of the benzo-diazepines on GABAA receptors (McDonald and Twyman,1992).The region of the nAChR a subunits that bears the binding

site for physostigmine, galanthamine, codeine and benzoqui-nonium has unique characteristics. In contrast to most of theN-terminal extracellular domain of the nAChR a subunits,the region between amino acids 118 and 137, which is part ofthe epitope region for FK1 and surrounds and includes the

amino acid Lys-125 (the residue that is affinity labeled by[3H]physostigmine) (Schroder et al., 1994), is amphipathic(Stroud et al., 1990). It has two hydrophilic residues, lysineitself and glutamate, and many hydrophobic residues, five ofwhich are aromatic amino acids. According to the modelproposed by Stroud et al. (1990), this region of the nAChR asubunits may have a b-pleated sheet conformation. Thus, ifone assumes that the residue Lys-125 is located at the bottomof a gorge, the two strings of amino acids lining the gorge willbe hydrophobic in essence with many p electron cloudsaround. This structure resembles that of the ACh bindingregion of AChE (Sussman et al., 1993) and may explain whymany AChE inhibitors can interact with the region includingand/or surrounding the residue Lys-125 of the nAChR asubunits. Because some studies have indicated that indola-mines, including the neurotransmitter 5-HT, can interact

Fig. 9. Potentiation of nicotinic responses by methylgalanthamine in PC12 cells. Top traces show that methylgalanthamine (methyl-GAL) by itselfis unable to evoke whole-cell currents when applied to PC12 cells, where it increases the peak amplitude of whole-cell currents evoked byapplication of a nonsaturating concentration of ACh (100 mM) to the cells. In the presence of methyl-GAL, the peak amplitude of the nicotiniccurrents evoked by ACh (100 mM) was similar to that of the currents evoked by the saturating concentration of ACh (1 mM) in the absence ofmethyl-GAL. Note, however, that in the presence of methyl-GAL, the nicotinic current evoked by ACh (100 mM) does not desensitize as much asdoes the current recorded by the saturating concentration of ACh, although both currents had about the same magnitude. (bottom graph)Histogram of the distribution of the amplitude of nicotinic currents evoked by application to various PC12 cells of ACh alone or by ACh in thepresence of methyl-GAL. Note that in the presence of methyl-GAL, the currents have larger amplitudes than in the absence of the drug. Holdingpotential, 260 mV.


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with the AChE found in the plaques of patients with Alzhei-mer’s disease (Wright et al., 1993), and given the apparentcorrelation between the ability of some anti-AChE com-pounds to bind to the active site of AChE and to the novelnAChR binding site, 5-HT was tested for its ability to mod-ulate ACh-evoked responses in PC12 cells. Of interest, 5-HTwas shown to mimic the potentiating action of galanthamineon ACh-evoked currents in PC12 cells (Schrattenholz et al.,1996). This result and the previous finding that the opioid

codeine, which is structurally related to galanthamine, canactivate nAChR channels via the same mechanism as phy-sostigmine suggest that 5-HT and, by inference, endogenousopiates could act as endogenous allosteric modulators of thenAChR function by binding to this novel nAChR site.There is increasing evidence that a given substance can

control synaptic activity in the brain by acting as the primaryagonist in one neurotransmitter system and as a modulatorin another system. Glycine is a classical example of such anendogenous substance. Whereas in glycinergic synapses gly-cine activates glycine-gated channels, in the glutamatergicsystem glycine acts as a coagonist at the NMDA-receptorchannels. Considering our findings, 5-HT may act as a fullagonist in serotoninergic synapses and as a neuromodulatorof the cholinergic neurotransmission involving nAChRs. De-velopment of novel therapeutics, therefore, should take intoaccount that the CNS function, in addition to being controlledby a neuronal network established by the neuronal wiring,could also be controlled by a chemical network established bythe dual action of a single substance acting as a neurotrans-mitter in one system and as a neuromodulator in anothersystem. This concept becomes very important when dealingwith drugs that act on neuronal nAChRs, particularly thosecomposed of a7 subunit, because it indicates that the func-tion of these receptors could be modulated indirectly by al-terations of the functions of neurotransmitter systems otherthan the cholinergic system itself.

Distribution of Functional nAChRs on theSurface of Hippocampal Neurons

In our electrophysiological studies, we have provided evi-dence that a single hippocampal neuron can express morethan one nAChR subtype (see fig. 3) (Alkondon and Albu-querque, 1993; Alkondon et al., 1994). However, there havebeen no studies dealing with the distribution of the differentnAChR subtypes on the surface of the hippocampal neurons.The issue of receptor distribution on the neuronal surface

becomes critical because the physiological role of a givenreceptor may be determined by its location on the neuronalsurface. For instance, it has been shown that upon high-frequency stimulation of presynaptic fibers, NMDA receptorsare activated postsynaptically, resulting in a APV-sensitiveincrease in intracellular Ca11 levels, which is confined to theactivated distal dendritic regions (Regehr and Tank, 1990).The same high-frequency stimuli can induce an APV-insen-sitive increase in intracellular Ca11 in proximal dendriticregions, which indicates that Ca11-permeable channelsother than the NMDA receptor channels are expressed in theproximal areas of the dendrites of hippocampal neurons andcontrol Ca11 entry in these cell compartments (Regehr andTank, 1990). By means of immunofluorescence, L-type Ca11

Fig. 10. Potentiation by galanthamine of (1)-anatoxin-a (AnTX)-evokedcurrents in hippocampal neurons is sensitive to blockade by FK1. (A)Galanthamine (GAL) increases the peak amplitude of whole-cell cur-rents evoked by application of a nonsaturating concentration of AnTX(10 mM) to hippocampal neurons in culture. In the presence of FK1,however, the effect of methyl-GAL is blocked. (B) Quantification of theeffect of GAL on AnTX-evoked type IA currents in hippocampal neuronsand of its sensitivity to blockade by FK1. (C) Note that in the presenceof GAL, AnTX-evoked type IA current does not desensitize as much asdoes the current recorded in the absence of GAL.


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channels have been shown to be clustered on the cell bodyand on the base of major dendrites of hippocampal neurons(Westenbroek et al., 1990). These findings support the con-cept that segregation of these Ca11-permeable channels onthe neuronal surface is critical for integration and processingof a synaptic input to the neurons. Although the NMDAreceptors, being at high density on distal dendritic regions,may serve a direct role in the induction of LTP at activatedsynapses, the L-type Ca11 channels, being at high density onthe cell body and proximal dendritic areas, may mediateintracellular regulatory events in the cell body in response to

the same synaptic inputs that lead to LTP at the distaldendritic areas of hippocampal neurons.We have addressed the distribution of nAChRs on the

surface of hippocampal neurons by recordings of whole-cellcurrents evoked by focal application of ACh to well-definedareas on the surface of hippocampal neurons. The set-upused in this study consisted of 1) a motor-operated uprightmicroscope that could be moved independently from the prep-aration-bearing stage, 2) an infrared filter, an infrared cam-era and an image processor that enhanced the contrast of theimage of the neurons allowing for estimation of distances

Fig. 11. Mapping of nAChRs on the somatodendritic domains of hippocampal neurons. (left panel) Infrared image of a hippocampal pyramidalneuron in culture is shown. (middle panels) Frames of images illustrate the positions of the agonist-delivery pipette at different regions of the sameneuron. (right panels) Traces of currents (type IA) induced by a 25-msec pressure application of ACh (3 mM) to the neuronal areas shown in themiddle panels are shown. Holding potential, 256 mV.


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with a precision of 0.5 to 1 mm and for visualization of minuteareas of the neuronal surface, including areas rich in den-dritic spines, 3) a computer-driven system that controlled themovements of the micromanipulators bearing the recordingpipette and the drug-delivery pipette and made it possible tofocally apply agonists of specific receptors to well-definedregions on the neuronal surface, and 4) a pressure-ejectionunit that was used to release the agonist onto the surface ofthe neurons (Alkondon et al., 1996a).The pipettes used to apply agonists to the neurons had tip

diameters ,1 mm to prevent receptor desensitization by ag-onist leak from the pipette tip, which can preclude the detec-tion of the fast-desensitizing, type IA currents and other fastdesensitizing responses such as GABA-activated Cl2 cur-rents. Also, the flow of the bath perfusion was directed oppo-site to the flow of the agonist solution from the agonist-delivery pipette. Maximal activation of the receptors locatedon the area covered by the agonist solution was achievedwhen the agonist-delivery pipette was positioned at 2 mmfrom the border of the neuronal surface, the duration ofagonist application was about 15 ms and the pressure to ejectthe agonist from the pipette was 20 pSi. Keeping the distanceof the pipette from the border of the neuronal surface fixed at2 mm, and the parameters for agonist application to the cellsfixed at 15-msec duration and 20-pSi pressure ejection, theagonist solution reached a forward distance of about 15 mm andcovered a lateral distance of 15 mm (Alkondon et al., 1996a).This advantageous technique is unique because it allows

for the investigation of the distribution of pharmacologicallyand kinetically identified functional receptors over the neu-ronal surface. However, it has some drawbacks. Given thatthe responses to agonists are recorded from the cell body andthe currents are evoked by agonist application to remoteareas of the neuronal surface, part of the currents may befiltered by the cable properties, and the peak current ampli-tude may be underestimated. Thus, our studies were limitedto regions within 60 mm from the recording pipette, distanceat which contribution of cable filtering was negligible asdetermined by the analysis of the relationship between risetime and peak amplitude of currents evoked by application ofthe agonists to different parts of the neurons (Alkondon et al.,1996a). Application of ACh (3 mM) in the presence of atropine(1 mM) to well-defined areas of the majority of the neuronsresulted in activation of fast-desensitizing, MLA-sensitive, typeIA current. In these neurons, application of ACh to the dendriticextensions resulted in the activation of type IA currents whoseamplitudes were smaller than those of the currents evoked byapplication of ACh to the cell body (fig. 11).Considering that the peak amplitude of whole-cell currents

evoked by activation of a single receptor subtype is propor-tional to the number of individual receptors activated by theagonist, an estimate of the current density at different areasof the neuronal surface can provide important informationabout the receptor density distribution in such neuronal ar-eas. Plots of the current density (estimated as the currentamplitude recorded from the soma/membrane area exposedto the agonist, i.e., pA/mm2) against the distance from the somaat which the agonist was applied revealed that the density oftype IA currents is substantially higher on the apical andbasal dendrites of pyramidal neurons and on the dendrites ofbipolar neurons than on the soma of these neurons (fig. 12).Considering that the same single-channel conductance ac-

counts for type IA currents evoked at the soma or at thedendrites, it is clear that the density of a7-bearing nAChRs ishigher on the dendrites than on the cell body of the neurons.The same analysis of type II currents evoked by applicationof ACh to various areas of the surface of hippocampal neu-rons and recorded from the cell body provided evidence thata4b2 nAChRs are also at higher density on the dendritesthan on the soma of these neurons. Analysis of the type IAand type II currents generated at more remote dendriticareas (up to 60 mm from the center of the soma) indicatedthat the current density in dendritic areas increases with thedistance from the center of the soma (fig. 12). These findingsare in agreement with previous immunocytochemical studiesof the specific binding of the nAChR-specific monoclonal an-tibodies FK1 and WF6 to hippocampal neurons, which re-vealed that spots of high-density immunolabeling, indicativeof synaptic regions, could be found along the dendrites(Schroder, 1992; Pereira et al., 1993a).Although the NMDA receptors and the a7 nAChRs are

apparently located on similar areas of the hippocampal neu-rons and have a high Ca11 permeability that can account fora sizable increase in Ca11 influx into the neurons, it is likely

Fig. 12. Relative distribution of nAChRs that give rise to type IA andtype II currents in hippocampal neurons in culture. Histograms showthe distribution of the current density (pA/mm2), which is directly pro-portional to receptor density, at various neuronal areas. The currentdensity (pA/mm2) at dendritic segments was normalized to that at thesoma.


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that these receptors have nonoverlaping roles in controllingchanges in intracellular levels of Ca11, because of the inwardrectification of a7 nAChRs-mediated currents and the out-ward rectification of NMDA-induced currents. At positivemembrane potentials, a7 nAChRs may not be functionalbecause of their blockade by intracellular Mg11 (see fig. 1)(Alkondon et al., 1994; Bonfante-Cabarcas et al., 1996),whereas NMDA receptors are fully operational. In contrast,at negative membrane potentials, a7 nAChRs would be fullyoperational, whereas NMDA receptors would be blocked byextracellular Mg11 (Nowak et al., 1984; Mayer and West-brook, 1987). Thus, one could expect some synaptic integra-tion to take place in dendritic spines that express bothNMDA receptors and a7 nAChRs. For instance, it is possiblethat activation of the a7 nAChRs by ACh (or choline), similarto activation of the AMPA-type glutamate receptors, couldlead to a local depolarization of sufficient magnitude to re-move Mg11 from the NMDA receptor rendering this receptorfully activatable. It is also feasible that increase in the intra-cellular Ca11 levels caused by Ca11 entry into the neuronsby activation of a7 nAChRs modulates the activation of theNMDA receptors. It should be emphasized that Ca11 influxinto dendrites plays a critical role in the induction of LTP inhippocampal neurons (Christie et al., 1996).

The Physiological Relevance of FunctionalnAChRs in Synaptic Modulation in the

Mammalian CNS: Studies of Neurons AcutelyDissociated from Different Areas of the

Human and the Rat BrainThe physiological relevance of functional nAChRs in CNS

neurons is still the subject of extensive investigation. Much ofthe knowledge available regarding the possible functions ofthe discrete subtypes of neuronal nAChRs is based on indi-rect clues obtained from behavioral studies of agonists andantagonists of the many CNS nAChR subtypes.It has been demonstrated that in rats, (2)-lobeline, (2)-

nicotine and (2)-cytisine (Haroutunian et al., 1985; Deckeret al., 1993) can improve retention test performance andwater maze deficits produced by septal lesions. Likewise,systemic administration of the nicotinic agonists anabasine,anabaseine and anabaseine derivatives to rats can improvemany memory tasks (Meyer et al., 1994). The ability of nic-otinic ligands to displace [3H]cytisine binding has been asso-ciated with their specific binding to the a4b2 nAChR sub-type, whereas the ability of such ligands to displacea-[125I]BGT binding has been associated with their specificbinding to the a7 nAChR subtype (Happe et al., 1994; Bar-rantes et al., 1995). The rank order of potency for nicotine,anabasine, anabaseine, and anabaseine derivatives in im-proving cognition does not completely match the rank orderof potency for these compounds in displacing the binding of(2)-cytisine, which suggests that at least two different sub-types of neuronal nAChRs are involved in memory acquisi-tion (Meyer et al., 1994). In fact, anabaseine and its deriva-tives have been shown to be selective agonists of a7-containing nAChRs (Briggs et al., 1995) and to facilitateinduction of LTP (Hunter et al., 1994). Thus, it is likely thatboth a4b2- and a7-bearing nAChRs play important roles incognitive functions. Initial studies have shown that DHbE, aspecific competitive antagonist at the a4b2 nAChR, impaired

water maze performances when administered intracere-broventricularly in rats, whereas MLA, a specific competitiveantagonist of ACh at the a7-containing nAChRs, produced atransient improvement of the performance of rats to find thehidden platform in the water maze test (Curzon et al., 1994).These findings suggested that acquisition of spatial informa-tion is modulated by distinct nAChR subtypes. However, noadditional information is available regarding the effects ofselective nicotinic antagonists on other learning and memorytasks.Anxiolytic effects of systemic administration of (2)-nico-

tine and other nicotinic agonists have been observed in lab-oratory animals and in human beings (Pomerleau, 1986).These effects are not common to all nicotinic agonists, giventhat systemic administration of cytisine, epibatidine,anabasine, anabaseine and its derivatives to rats has noeffects on anxiety. Thus, it is likely that the anxiolytic effectsof some nicotinic agonists are accounted for by the actions ofthese agonists on a specific subtype of neuronal nAChR. Oftherapeutic interest, the anxiolytic effects of nicotinic ago-nists, in contrast to those of benzodiazepines, are not accom-panied by cognitive deficits. Unfortunately, the anxiety fre-quently observed in patients with Alzheimer’s disease is stilltreated with benzodiazepines. The development of a nicotinicagonist with anxiolytic effects would be of great advantagefor these patients.(2)-Nicotine and many nicotinic agonists have an analge-

sic effect in animal species (Pomerleau, 1986; Badio andDaly, 1994). The findings that this effect can be prevented ifthe animals are pretreated with mecamylamine, but not withhexamethonium, indicates that this effect is mediated byCNS actions of the nicotinic agonists. It seems that antino-ciception by nicotinic agonists is associated with a specificsubtype of neuronal nAChR, because agonists that are moreselective for a4b2 neuronal nAChRs, e.g., nicotine and (6)-epibatidine, can induce analgesia when administered to miceand rats, whereas agonists that are more selective for thea7-bearing nAChRs, e.g., anabaseine and its derivatives, areineffective antinociceptives. Although the exact mechanismby which activation of neuronal nAChRs leads to analgesiaremains to be determined, cholinergic synaptic transmissionmediated by a4b2 nAChRs has been observed in brainstemslices containing both the nucleus ambiguus and the zonaintermedialis reticularis parvicellularis of the rostral me-dulla oblongata, which are part of the nociceptive pathways.Fast excitatory spontaneous postsynaptic potentials, whoseamplitude and frequency can be decreased markedly byDHbE, have been recorded from neurons of the nucleus am-biguus, hence suggesting that a4b2 nAChRs expressed inneurons of this nucleus are innervated by cholinergic neu-rons present in the slice (Zhang et al., 1993). Retrogradetracing of the afferents to the nucleus ambiguus combinedwith choline acetyltransferase immunocytochemistry re-vealed that the zona intermedialis reticularis parvicellularisof the medulla oblongata is the main source of cholinergicinput to the nucleus. In fact, electric stimulation of the zonaintermedialis reticularis parvicellularis resulted in activa-tion of excitatory postsynaptic potentials in neurons of thenucleus ambiguus that were very sensitive to blockade byDHbE (2–5 pmol); DHbE reversibly reduced the peak ampli-tude of the indirectly elicited postsynaptic potentials (Zhanget al., 1993).


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Because there is no evidence that a-BGT-sensitive, a7nAChRs mediate fast, excitatory synaptic transmission inthe CNS, it has been speculated that these receptors maymodulate synaptic function, neuronal signaling, and neuro-nal development. In fact, a7-containing neuronal nAChRshave been shown to control neurite outgrowth and excitotox-icity (Chan and Quik, 1993; Akaike et al., 1994; Pugh andBerg, 1994; Donnelly-Roberts et al., 1996). The high Ca11

permeability of the a7 nAChRs suggests that these receptorsmay be involved in many other neuronal functions that aredependent on the intracellular levels of Ca11.In addition to hippocampal neurons, neurons from the

olfactory bulb of rats express a7 nAChRs (Alkondon andAlbuquerque, 1994). Activation of these receptors results inelicitation of fast-desensitizing, a-BGT-sensitive currentsthat resemble type IA currents recorded from the hippocam-pal neurons (fig. 13). More interesting, however, is the factthat application of ACh or other nicotinic agonists to someolfactory bulb neurons in culture results in a substantialincrease in the frequency of spontaneous postsynaptic cur-rents (fig. 13) (Alkondon et al., 1996c). This increase in the

frequency of spontaneous postsynaptic currents is inhibitedreversibly by MLA (1 nM) and lasts for a few millisecondsafter the neurons are exposed to ACh (Fig. 13), which indi-cates that this response is mediated by a7 nAChRs. Usingantagonists of various ligand-gated receptors, it was possibleto demonstrate that the neurotransmitter accounting for thepostsynaptic currents whose frequency was increased by thenicotinic agonist is glutamate, and that the receptor mediat-ing such currents is the AMPA-type glutamate receptor (fig.13). Finally, because the ACh-induced increase in glutamaterelease is insensitive to TTX, the a7 nAChR should be locatedon the presynaptic side of glutamatergic synapses, wherebyits activation leads to modulation of transmitter release(Alkondon et al., 1996c). Thus, in olfactory bulb neurons ofthe rat, a7 nAChRs located on presynaptic glutamatergicterminals are capable of modulating the function of glutama-tergic synapses by controlling the release of glutamate (fig.13; Alkondon et al., 1996c). Presynaptic a7 nAChRs have alsobeen reported to control the release of glutamate in chickbrain neurons and in neurons of the CA3 field of the hip-pocampus (McGehee et al., 1995; Gray et al., 1996).Although neurons in culture maintain many of their native

properties, the expression of certain proteins can be affectedsubstantially by the composition of the medium used to cul-ture the neurons. Thus, to prove that functional nAChRs areindeed expressed in mammalian CNS neurons developed invivo, and that the characteristics of these receptors are notdifferent from those of the receptors expressed in culturedneurons, we have applied the patch-clamp technique to neu-rons dissociated from various areas of the rat brain.For many years, the use of acutely dissociated neurons has

been hampered by the lack of a reliable and reproduciblemethod that would allow for the dissociation of single neu-rons without excessive damage. Most of the studies directedat characterizing the properties of functional ligand-gatedion channels expressed in neurons of the central or the pe-ripheral nervous systems have relied on the use of proteolyticenzymes as dissociating agents. However, such enzymes al-ter the sensitivity of ligand-gated channels to their naturalagonists or to allosteric ligands. For instance, the NMDAreceptors of hippocampal neurons are inactivated by trypsin(Allen et al., 1988). Recent studies on single muscle fibershave also shown that the extracellular domain of nAChRs ishighly sensitive to enzymatic treatment (Nascimento et al.,1996). After proteolytic treatment of frog muscles for isola-tion of single muscle fibers, the frequency of ACh-evokedsingle-channel currents as well as the frequency of single-channel currents elicited by physostigmine-like compoundsdecrease with increasing exposure of the muscle fibers toproteases. If neuronal a7 nAChRs were as sensitive as mus-cle nAChRs to proteolytic treatment, one would expect to findsmall nicotinic responses in neurons enzymatically dissoci-ated from the hippocampus. Thus, to overcome the problem ofenzymatic treatment, an improved technique was developedin our laboratory that allows for the isolation from specificCNS areas of single viable neurons bearing many long den-drites (fig. 14) (Barbosa et al., 1996).Application of ACh or other nicotinic agonists to most

neurons mechanically dissociated from the CA1 field of thehippocampus of 3- to 25-day-old rats resulted in activation offast-desensitizing, MLA-sensitive nicotinic currents whosecharacteristics resembled those of type IA currents recorded

Fig. 13. Evidence for the presence of postsynaptic and presynaptica-BGT-sensitive nAChRs in olfactory bulb neurons. ACh-evokedwhole-cell current (Direct) that is blocked by MLA but not by CNQX ismediated by postsynaptic nAChRs. ACh-evoked whole-cell currents(Indirect) that are blocked by both MLA and CNQX represent activationof presynaptic nAChRs located on glutamatergic terminals.


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from hippocampal neurons in culture. Approximately 65% ofthe acutely dissociated hippocampal neurons responded toACh with type IA currents whose peak amplitude variedbetween 15 and 750 pA at the membrane potential of 250 mV(Barbosa et al., 1996). The average amplitude of the currentsrecorded from the hippocampal neurons dissociated mechan-ically according to this technique was larger than that of thecurrents recorded from mechanically dissociated hippocam-pal neurons bearing short dendrites (Ishihara et al., 1995;Barbosa et al., 1996). This discrepancy could be accounted forby the fact that a large number of nAChRs is found in thedendrites (Alkondon et al., 1996a).That the mechanically dissociated neurons are viable and

suitable for characterization of the elements participating insynaptic transmission was proven by the finding that spon-taneous postsynaptic currents could be recorded from manyof these neurons (see fig. 14), and that the frequency of thesecurrents could be altered by cations known to alter transmit-

ter release, e.g., Pb11 (Barbosa et al., 1996). Therefore, notonly do the mechanically dissociated neurons bear long den-dritic branches but they also retain functional synaptic ter-minals attached to them. The development of this powerfultechnique has allowed us to proceed forward into the studiesof the properties of functional ligand-gated channels ex-pressed in neurons from the human brain (Pereira et al.,1997).In an attempt to characterize the subtypes of ligand-gated

channels in neurons from the human brain aiming at defin-ing the effects of agonists, antagonists, and other modulatorsof receptor activity, particularly molecules that have provenuseful to treat and/or prevent a number of neuropathologicalconditions including epilepsy and Alzheimer’s disease, wehave applied the methodology of mechanic dissociation tosamples of lateral cortex removed during temporal lobectomyfrom epileptic patients. The specimens were taken from theroute of access to the damaged area of the brain of thepatients subjected to the neurosurgical procedure, and were,therefore, unlikely to be affected by the pathological condi-tion. Like the neurons acutely dissociated from the rat brain,neurons acutely dissociated from human neocortical speci-mens showed high frequency of spontaneous postsynapticcurrents, indicating that they also retained on their surfacefunctional synaptic terminals. In addition, these neuronsresponded to ACh (1 mM) with fast-desensitizing currentswhose characteristics resembled those of type IA currents.This constitutes the first direct evidence that functional a7-type nAChRs are indeed expressed in the human brain(Pereira et al., 1997).A substantial amount of information regarding the physi-

ological roles of some ligand-gated ion channels and voltage-gated ion channels has been gathered from studies on brainslices. Although in-depth analyses of the pharmacologicaland kinetic properties of such channels are made difficultbecause of the barriers for drug access to the cells in theslices, the use of this preparation has the advantage of al-lowing for the identification of receptor function within itsnatural environment. Thus, we have investigated the func-tions mediated by neuronal nAChRs expressed in neuronsvisualized in hippocampal slices.Hippocampal slices were obtained according to the proce-

dure briefly described (Alkondon et al., 1996b). After sacri-ficing Sprague-Dawley rats (3- to 30-day-old) under CO2 nar-cosis, the cerebral hemispheres were removed and placed incold physiological solution. After dissection, the hippocampiwere placed on the stage of a vibrating slicer (FTB, Wein-heim, Germany), and 200- to 300-mm transverse slices werecut in ice-cold ACSF solution, which consisted of (in mM):NaCl, 124; NaHCO3, 26; KCl, 3; Na2HPO4, 1.25; CaCl2, 2;MgCl2, 2; and glucose, 10. Slices were incubated in aeratedACSF at 32–35°C for 30 to 60 min and thereafter at roomtemperature.Visualization of individual neurons in the slices was made

possible by the use of infrared microscopy (fig. 15). The sur-face of the visualized neurons was cleaned by a gentle streamof ACSF to permit ready access of the patch-clamp pipetteand the drug-delivery pipette to the cell surface, and agonistwas delivered to the neurons by short pulses of positivepressure using a pico-injector (PLI-100; Medical SystemsCorp., Greenvale, NY). Under these conditions, application ofseveral nicotinic agonists, including ACh, to pyramidal neu-

Fig. 14. Characterization of ligand-gated channels in mechanicallydissociated neurons. Infrared images of rat (P15) CA1 hippocampalneurons that were acutely dissociated without the assistance of en-zymes. Type IA nicotinic current elicited by application of ACh to one ofthese neurons is shown at the bottom.


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rons visualized in the CA1 field of hippocampal slices evokedslowly decaying whole-cell currents that were accompaniedby an increased frequency of spontaneous postsynaptic cur-rents (fig. 15). The responses of the neurons to ACh werenicotinic in nature, because they were blocked by d-tubocu-rarine and were insensitive to blockade by atropine (fig. 15).The nAChR underlying the action of ACh in CA1 neurons isalso unlikely to contain the a7 subunit, because MLA isunable to inhibit the ACh-induced responses (Alkondon et al.,1996b). If the nAChR underlying this cholinergic functioncontains the a7 subunit, this subunit may be combined withother subunits that reduce the apparent potency of MLA ininhibiting the activation of the receptor. In the CA1 field ofthe hippocampus, it is unlikely that the nAChRs controllingtransmitter release are located in the presynaptic terminals,because TTX (200 nM) inhibited the ACh-induced increase infrequency of spontaneous postsynaptic currents, which indi-cates that depolarization resulting from activation of

nAChRs in presynaptic neurons leads to the generation ofaction potentials that propagate along the neuronal axon andinvade the presynaptic terminal causing the release of neu-rotransmitter. The kinetic and pharmacological analysis ofthe spontaneous postsynaptic currents whose frequency isincreased by nicotinic agonists indicates that those currentsare GABAergic; they are blocked by the GABAA receptorantagonist picrotoxin and have a slow decay phase, charac-teristic of GABA-mediated spontaneous postsynaptic cur-rents (Alkondon et al., 1996b). Thus, our studies in the slicesdemonstrate that functional nAChRs are expressed on CA1hippocampal neurons of GABAergic nature and that depolar-ization subsequent to the activation of these receptors in-creases the release of GABA.Our findings contrast with the findings of a recent study on

neurons visualized on the CA3 field of hippocampal slices inwhich evidence was provided that neuronal nAChRs are ex-pressed on presynaptic glutamatergic terminals, and that the

Fig. 15. Characterization of nicotinic responses in neurons visualized in hippocampal slices. Infrared images of rat (P15) CA1 hippocampal neuronsin a slice preparation is shown on the left. On the right are the whole-cell currents evoked by ACh and (1)-epibatidine [(1)Epi] in two such neurons.Bath application of d-tubocurarine (d-TC) abolished the responses elicited by ACh. Holding potential, 250 mV.


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activation of these receptors increased the release of gluta-mate (Gray et al., 1996). Apparently, an a7 nAChR is pre-dominantly found in presynaptic glutamatergic terminals ofthe CA3 field of the hippocampus, whereby its activationleads to an increase in glutamate release, whereas a func-tional nAChR, unlikely to bear the a7 subunit, is predomi-nantly expressed on GABAergic neurons in the CA1 fieldwhereby its activation leads to neuronal depolarization thatultimately accounts for GABA release (fig. 16). These find-ings are in good agreement with the concept that to developefficacious therapeutic agents capable of balancing the cho-linergic function whenever it is impaired, one has to unveilthe receptor subtypes involved in the neuronal functionsaltered in a given pathological condition.

Concluding RemarksnAChRs are believed to be intimately involved in learning

and memory. The level of expression of neuronal nAChRs isreduced in the brain of patients with Alzheimer’s (Schroder etal., 1989, 1991, 1996), and we and others have providedevidence that functional nAChRs and glutamate receptorsare expressed in hippocampal and cortical neurons. There-fore, it is tempting to speculate on ways by which nAChRsmay participate in LTP, the best studied model for memoryacquisition.LTP is induced rapidly by small bursts of activity, and it is

characterized by increased efficacy of neurotransmission. Inaddition, some forms of LTP, particularly the NMDA-depen-dent LTP, are consistent with Hebbian learning mechanisms(Hebb, 1949). In NMDA-dependent LTP, the postsynapticNMDA receptor channel opens in response to presynaptically

released glutamate only if the postsynaptic cell is sufficientlydepolarized. On activation of the NMDA receptors, Ca11

enters the neurons. Elevation of the intracellular levels ofCa11 can result in activation of intracellular kinases, whichmodify the postsynaptic receptors increasing their sensitivityto glutamate, and/or in the release of retrograde factors that,diffusing to the presynaptic terminal, can increase transmit-ter release.Considering the NMDA-dependent LTP, activation of

postsynaptic nAChRs, like the activation of AMPA-type glu-tamate receptors, could provide the level of postsynaptic de-polarization that is a prerequisite for NMDA receptor gatingby glutamate. Thus, coincidental activation of both a cholin-ergic and a glutamatergic terminal impinging on the samespine could result in strengthening of synaptic transmissiontranslated as LTP. In the case that the postsynaptic nAChRwere of the a7 subtype, activation of this channel could causea significant elevation of the cytosolic concentrations of Ca11

to the levels necessary for release of retrograde messengerssuch as arachidonic acid, NO and CO, or for activation of thesecond messenger cascades known to play a role in the mod-ulation of LTP. Ultimately, given that a7 nAChRs present inpresynaptic terminals can control transmitter release fromhippocampal neurons, in part because of their high Ca11

permeability, and that these receptors can be activated bycholine, the hydrolysis product of acetylcholine, it is possiblethat choline could serve as a retrograde messenger that per-mits sustained release of neurotransmitter. In this scenario,if we consider a spine that expresses both a cholinergic re-ceptor (upon which synapses a cholinergic terminal) and theNMDA receptor (upon which impinges a glutamatergic ter-

Fig. 16. Hypothetical model showing modulation of synaptic activity by neuronal nAChRs. Different color codes for the nAChRs represent differentreceptor subtypes. The nAChR shown on the presynaptic axon represents any a-BGT-insensitive nAChR subtype, whereas the nAChR shown onthe postsynaptic neuron represents any nAChR subtype.


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minal bearing the a7 nAChR), coincidental activation of bothterminals would result in the release of ACh and glutamate,each of which would activate its own postsynaptic receptorunder the proper conditions. Then, hydrolysis of ACh byAChE would generate choline, which by diffusion could reachthe a7 nAChR located on the presynaptic glutamatergic ter-minal leading to a sustained release of glutamate, even afterstimulation of the presynaptic neuron was terminated. Thiscould represent a mechanism by which ACh acts within thesynapse in which it is released, and choline acts in the sameor neighboring synapses.It is noteworthy that the a7 nAChR belongs to the evolu-

tionary oldest group of nAChRs (LeNovere and Changeux,1995). It appears feasible that originally choline rather thanACh was the natural transmitter for this nAChR and thatthe evolution to ACh was in part caused by the need to havea “two-step transmitter,” i.e., a rapidly acting, rapidly inac-tivatable one (ACh) and a more slowly removed one (choline).Interestingly, ACh is the only neurotransmitter known to berapidly inactivated by a specific enzyme, whereas the actionof other neurotransmitters is terminated by transport intoneighboring cells or by receptor desensitization.

Acknowledgments

The authors are grateful to the excellent technical assistance ofMs. Mabel A. Zelle, Ms. Barbara Marrow and Mr. Benjamin Cum-mings. The graphical assistance of Mr. Thomas Jemski from theIllustrative Services of the University of Maryland is also gratefullyacknowledged.

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Properties of Neuronal Nicotinic Acetylcholine Receptors