Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system

Review Article

Muscarinic toxins: novel pharmacological toolsfor the muscarinic cholinergic system

D. Jerusalinskya,b,*, E. Kornisiuka, P. Alfaroa, J. Quillfeldtc,A. Ferreirac, Verde E. Riala, R. DuraÂ nd, C. CervenÄ anskyd

aInstituto de BiologõÂa Celular y Neurociencias ``Profesor Eduardo De Robertis'', Facultad de Medicina,

Universidad de Buenos Aires, Paraguay 2155, 3er piso, 1121, Buenos Aires, ArgentinabCido BaÂsico ComuÂn, Universidad de Buenos Aires, Paraguay 2155, 3er piso, 1121, Buenos Aires,

ArgentinacDept. Bio®sica, Universidad Federal Do Rio Grande Do Sul (Campus), Porto Alegre, RGS, Brazil

dInstituto de Investigaciones BioloÂgicas ``Clemente Estable'', Montevideo, Uruguay

Received 1 August 1999; accepted 11 August 1999

Abstract

Muscarinic receptors are widely spread throughout the body, and are involved in theregulation of fundamental physiological processes, like the modulation of the heart rate,control of motor systems and modulation of learning and memory. In the central nervous

system the cholinergic transmission is mainly mediated by muscarinic receptors; there are®ve subtypes that are all expressed in the brain of mammals (m1±m5). There are regionaldi�erences in their concentrations in the brain and more than one subtype is expressed inthe same cell. It has been di�cult to study their localization and function in vivo due to the

lack of ligands that exclusively act on one subtype of the receptor. We studied the action ofthe muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspisangusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3H±NMS

as reporter of the inhibition of its own binding, to either native or cloned receptors. Wehave also studied the in vivo e�ects on memory retention of the injection of the toxins intodiscrete brain regions. The muscarinic toxins appear to be invaluable tools to study

receptor pharmacology, physiology and structure/function relationships. They would enablethe design of new, more selective, pharmacological agents. # 2000 Published by ElsevierScience Ltd. All rights reserved.

0041-0101/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.

PII: S0041 -0101 (99)00196 -8

Toxicon 38 (2000) 747±761

www.elsevier.com/locate/toxicon

* Corresponding author. Fax: +54-11-4508-3675.

1. Introduction

1.1. Acetylcholine as neurotransmitter

Acetylcholine (ACh) was the ®rst to be identi®ed as a central neurotransmitterand until about 50 years ago it was the only one recognized in the brain. At thosetimes, it was thought that this was used by all chemical synapses. Now we knowmore than 50 neurotransmitters plus neuromodulators, and some putativeneurotransmitters are still waiting to get onto the list! However, ACh is one of themajor neurotransmitters, subserving many relevant functions, both in the centraland peripheral nervous system.

1.2. ACh receptors

1.2.1. Muscarinic ACh receptorsOne neurotransmitter may have di�erent e�ects either at the same receptors or

at di�erent receptors, located in a di�erent, or even the same, neuron. Based uponpharmacological evidence, that showed di�erent e�ects of ACh at di�erent tissues,it was proposed that there are two main families of ACh receptors: nicotinic andmuscarinic, because those ACh e�ects were mimicked by the natural alkaloidsnicotine and muscarine, respectively (Dale, 1914; Weiner and Taylor, 1985). Thiswas later con®rmed by the molecular biology of the receptors. In particular,muscarinic ACh receptors (MAChR) are widely spread throughout the body, andare involved in the regulation of many fundamental physiological processes, suchas gut and other smooth muscle contractions, modulation of heart rate, control ofmotor systems, and the modulation of attention, learning and memory.

In the central nervous system there is only one group of basal neuronsprojecting to the cerebral cortex or to the hippocampus, which is capable ofsynthesizing and releasing ACh and the neurotransmission in those target regionsis mainly mediated by MAChR. They belong to the seven-transmembranespanning protein receptors whose response is mediated by the membrane proteinsthat bind GTP, named G-proteins (Nathanson, 1987; Hulme et al., 1991). TheMAChR are located either in the pre- or postsynaptic membrane, in cholinergicor other neurons, and ®ve subtypes are expressed in the brain of mammals (m1±m5). There are di�erences in concentration of receptor subtypes in various regionsof the brain, but often, more than one subtype is expressed in the same cell. Then,how to study muscarinic receptors? Although we know about the existence of the®ve molecules that constitute MAChR (m1±m5), their coding genes that havebeen cloned (Kubo et al., 1986; Bonner et al., 1987; Hulme et al., 1990, 1991) andthe corresponding mRNA, it has been extremely di�cult to study theirlocalization and function in vivo due to the lack of selective ligands thatexclusively act on one subtype of the receptor. Pirenzepine, reported as a relativeselective antagonist for m1 MAChR (Hammer and Giachetti, 1982), also bindswith rather high (almost same) a�nity to the m4 subtype and with slightly lowera�nity to the m3 and m5 subtypes (see Caul®eld, 1993; Kornisiuk et al., 1999).

D. Jerusalinsky et al. / Toxicon 38 (2000) 747±761748

There is a high correlation in pharmacological characteristics of the di�erentMAChR subtypes of various species, i.e. human (DoÈ rje et al., 1991), rabbit andrat (Lazareno et al., 1990).

The speci®c antibodies have been shown to be useful to study proteinlocalization and, sometimes, protein function. Some antibodies have beendeveloped against MAChR subtypes (Levey, 1993). The more speci®c antibodiesare usually directed against the less homologue region of the receptor, whichbelongs to the intracytoplasmic domain. Thus, they are not useful to reach andblock the receptors from the extracellular side, to study their functions, since theagonist site is likely to be located in a pocket, formed by the transmembranedomains, near the external surface of the membrane (Jones et al., 1992; Hulme etal., 1995; Rouse et al., 1998; Van Der Zee and Luiten, 1999).

1.3. The history of muscarinic toxins

In the early 80's some e�ects of the venom of Dendroaspis angusticeps (EasternGreen Mamba) suggested that it contained some cholinomimetic component(Harvey and Karlsson, 1980). Subsequently, the fasciculins, which behaved asinhibitors of acetylcholinesterase activity (RodrõÂ guez-Ithurralde et al., 1983) anddendrotoxins, blockers of K+ channels (Harvey and Karlsson, 1984) wereisolated. Adem et al. (1988) assayed two fractions of proteins, MT1 and MT2(named muscarinic toxins MTs), that were able to inhibit the binding of themuscarinic antagonist 3H±QNB to cerebral cortex. Since the radioligand bindingwas only partially decreased (50%), in the presence of either MT1 or MT2, it wassuggested that those proteins would bind to only some subtypes of the receptor.

Following similar procedures as those to isolate fasciculin and dendrotoxin, weisolated two proteins with muscarinic binding properties from the same venom.Later on it was shown that those proteins were the same as MT1 and MT2(Jerusalinsky et al., 1989, 1992).

Karlsson and colleagues have continued isolating di�erent proteins of closerelated molecular weight (around 7 kDa), with muscarinic actions (Jolkkonen etal., 1993). Some of them have been sequenced, as MT1, MT2, MT3 (same as m4-toxin) (Ducancel et al., 1991; Karlsson et al., 1991; Max et al., 1993a,b; Liang etal., 1996), MT4 (Vandermeers et al., 1995) and MT7 (Jolkkonen, 1996) anisotoxin of m1-toxin (Max et al., 1993a; Potter et al., 1996). All of them are rathersimilar in the position of their eight cysteine residues. This implies that theyshould have a similar pattern of disul®de bonds and hence, a similar three-dimensional structure. The ®rst structure to be determined was that of MT2,through nuclear magnetic resonance and molecular modelling (SeÂ galas et al.,1995). This belongs to the group of the three-®ngered toxins, with three loops richin b-sheet as secondary structure, linked to a globular core with the four disul®debonds.

There are 13 conserved amino acids in the curaremimetic a-neurotoxins fromthese venoms (short chain a-neurotoxins) that were postulated to be the site to

D. Jerusalinsky et al. / Toxicon 38 (2000) 747±761 749

bind to the nicotinic receptor. Only two of those amino acids are in the sequenceof MT1 or MT2 (Ducancel et al., 1991; Karlsson et al., 1991).

We have studied the action of MT1, MT2 and MT3 on MAChR by using theclassical muscarinic radioligand 3H±NMS as reporter of the inhibition of itsbinding, to either native or cloned receptors in membranes (Jerusalinsky et al.,1989, 1992; Kornisiuk et al., 1995a,b) and in brain slices (Jerusalinsky andHarvey, 1994; Jerusalinsky et al., 1998). We have also studied the in vivo e�ects ofthe injection of the toxins into discrete brain regions, on the retention of memory(Jerusalinsky et al., 1993, 1995, 1998). We will also comment on somecollaborative experiments we have performed on the e�ects of the toxins onisolated organs like rabbit vas deferens (Jerusalinsky and Harvey, 1994; Kornisiuket al., 1995a,b; Harvey et al., 1997).

2. Methods and results

2.1. Binding experiments in membranes with cloned muscarinic receptors

2.1.1. Materials and methodsThe radioligand binding assays were carried out in duplicate in aliquots of

membranes containing 0.02 mg protein/ml of m1, m2, m3, m4 or m5 clonedhuman MAChR subtypes (BioSignal Co) in 50 mM Tris±HCl bu�er, with 10 mMMgCl2 and 1 mM EDTA, pH 7.4 (Jerusalinsky et al., 1992, 1995; Jerusalinsky andHarvey, 1994; Kornisiuk et al., 1995b).

The inhibition curves were performed with di�erent concentrations of MTs(0.001±15 mM) against a ®xed concentration of 0.6 nM of the classical muscarinicradioactive ligand 3H±NMS (NEN), for 60 min at 258C. The reaction was

Fig. 1. Inhibition curves of the binding of 3H±NMS (0.6 nM) to human cloned MAChR of the m1 and

m4 subtypes, by MT1 (A), MT2 (B) or MT3 (C). The concentration of MTs were in the range 0.001±

15 mM.


terminated by ®ltering through Whatman GF/B ®lters. To determine non-speci®cbinding, similar aliquots with 50 mM atropine were simultaneously incubated. The®lters were washed three times with cold bu�er, dried and put into a vialcontaining scintillation ¯uid (for details, see Jerusalinsky et al., 1992; Kornisiuk etal., 1995a,b). The speci®c binding was obtained by subtracting the non-speci®cfrom the total binding. The plots and calculated parameters have been obtainedusing the Graph Pad Prism software, with non-linear regression to adjust theinhibition data.

2.1.2. ResultsThe muscarinic toxins inhibited the binding of the non-selective antagonist S3H-

QNB and 3H±NMS, to native (Adem et al., 1988; Jerusalinsky et al., 1989, 1992,1994, 1995; Jolkkonen et al., 1993, 1995) and cloned MAChR (Jerusalinsky andHarvey, 1994; Kornisiuk et al., 1995b; 1997; Jerusalinsky et al., 1998). MT1, MT2and MT3 exhibit di�erent inhibition binding pro®les with respect to the di�erentMAChR subtypes. The inhibition curves to m1 and m4 cloned receptors areshown in Fig. 1 (A, B, C). MT1 appears highly selective for m1 and m4 subtypes,with almost the same a�nity (Ki at m1, 22 nM; Ki at m4, 29 nM). MT2 exhibitshigher selectivity for m1 compared to m4, although its a�nity is lower than thatof MT1 (Ki at m1, 632 nM; Ki at m4, 1.89 mM). MT3 shows very high a�nity form4 (Ki, 1.38 nM) and 214 fold lower a�nity for m1 receptor. These resultscorrespond to one experimental inhibition curve out of three.

All the three MTs show rather low a�nity or even have no e�ects on thebinding of 3H±NMS at m2, m3 or m5 receptor subtypes. While MT1 and MT2have little a�nity for m5 and do not seem to bind to m2 or m3 receptors(Kornisiuk et al., 1995b), MT3 shows very little a�nity for all the subtypes exceptm4 (i.e., Ki=1.1 mM at m1 receptor, compared to 1.38 nM at m4) (see Liang etal., 1996; Jerusalinsky et al., 1998).

2.2. Distribution of muscarinic toxins sites in brain slices

2.2.1. Materials and methodsAdult male Wistar rats (200±250 g body weight) were used to obtain frozen

sections of the brain. The animals were mantained on a 10 light/14 dark h cyclehaving food and water ad libitum. The rats were decapitated, the brains wererapidly removed, cooled to ÿ208C and then kept frozen at ÿ708C until used.Sagital frozen sections (15 mm thick) 1.9 mm from Bregma (Paxinos and Watson,1986) were cut using a cryostat, thaw mounted into glass slides and stored atÿ708C until used.

Frozen brain sections were warmed to 48C, preincubated for 15 min at 48C in50 mM Na+, K+, PO3ÿ

4 bu�er, pH 7.4, with 0.01% bovine serum albumin (BSA),and then incubated for 30 min at 208C in fresh bu�er in the presence of increasingconcentrations of either MT1, MT2 or MT3 (0.005±10.00 mM). A concentrationof 0.5 nM 3H±NMS was then added and the incubation continued for 60 min.Other brain sections were simultaneously incubated with the muscarinic antagonist


atropine (10 mM) and the same concentration of 3H±NMS, to determine the non-speci®c binding, which was always less than 10% of the total. After theincubation, slides were rinsed three times (15 s each) in bu�er at 48C, followed bya dip in ice-cold destilled and deionized water.

Sections were dried with cold air and exposed to tritium-sensitive Hyper®lm(Amersham) in light tight X-ray cassettes. Standars ([3H]-microscales, Amersham)of known radioactivity were co-exposed. After 3 weeks the ®lm was developed inDektol developer (Kodak).

Autoradiograms were analyzed by computed assisted densitometry (MCIDSystems, Imaging Research, St. Catherines, Ont., Canada). Film optical densitywas converted to tissue radioactivity concentration using a regression curvederived from the radioactive co-exposed standards.

2.2.2. ResultsWe selected only MT2 and MT3 for these assays by virtue of their de®ned

selectivity for one receptor subtype. Quantitative experiments performed withMT1 are in progress and are more di�cult to interpret since MT1 binds to m1and m4 receptors with rather similar a�nities.

Inhibition curves of the binding of 3H±NMS to rat brain slices are shown inFig. 2A and B. In the Fig. 2A it is shown the inhibition by MT2 in the striatum,frontal cortex, and CA1, CA3 and dentate gyrus from the hippocampal

Fig. 2. Inhibition curves of the binding of 3H±NMS (0.5 nM) to rat brain slices by MT2 (A) and MT3

(B). The data were obtained by densitometric quanti®cation of autoradiograms analyzed by computed

assisted densitometry. Optical density was converted to tissue radioactivity concentration by the

regression curve from the radioactive co-exposed standards. The inhibition is expressed as a percentage

of the total binding. The data were adjusted using non-linear regression. The concentration of MTs

were in the range 0.005±15 mM.


formation. Fig. 2B is the plot of similar assays performed with MT3 in the samebrain regions.

The highest level of inhibition by MT2 was reached in the dentate gyrus (76%).The a�nities were not statistically di�erent between the dentate gyrus and thefrontal cortex (Ki 3.6 mM vs Ki 2.6 mM). The lowest inhibition was achieved in thestriatum (24.8%), where it was not possible to get any good ®tness. This wasfollowed by the frontal cortex (43.6% inhibition, Ki 2.6 mM), the CA3 region ofthe hippocampus (inhibition of 48%, Ki 1 mM) and then CA1 (52.8%; Ki 4 mM)(Fig. 2A). The inhibition curves for the dentate gyrus and for CA1 ®tted to asimple competition model.

Although a high level of inhibition of 3H±NMS binding by MT3 was achievedin the striatum (Fig. 2B), which would be the structure the richest in m4 receptors,the maximal inhibitions reached both in the striatum and the dentate gyrus weresimilar (89%), while the a�nity in the striatum (Ki 368 nM) was about 1.5 foldthat in the dentate gyrus (Ki 560 nM). In the CA3 region, MT3 reached 65%maximal inhibition (Ki 231 nM), while the CA1 region and the frontal cortexshowed similar total inhibition (69%). The a�nity in CA1 was almost half that inthe frontal cortex (Ki in CA1, 843 nM; Ki in frontal cortex, 493 nM). Theinhibition curves for all the structures analysed ®tted to a simple competitionmodel.

The binding of 3H±NMS inhibited by either MT2 or MT3 could be taken as anindication of the corresponding concentrations of m1 and m4 receptorsrespectively.

2.3. Behavioral experiments

2.3.1. Materials and methodsAdult male Wistar rats weighing 200±250 g were bilaterally implanted, under

thionembutal anesthesia (30 mg/kg), with a 27-gauge guide cannulas aimed at thedorsal hippocampus, following the coordinates from the Atlas of Paxinos andWatson (1986) using a stereotaxic apparatus for small animals (David KopfInstr.). A group of rats were trained for a step-down inhibitory avoidance task inone session, 4 to 5 days after surgery. The apparatus consisted in a50 � 24 � 25 cm acrylic box with a ¯oor of parallel bronze bars. A 5 cm high,7 cm wide isolated platform was on the left side.

In the training session the animal was placed on the platform; on steppingdown with its four paws on the gride, the rat received a 0.5 mA, 4 s foot-shock,after which it was taken out from the training box and kept in the home-box.Twenty four h later the test session took place. The time the animal spent on theplatform in the training session was the training latency. In the test session, nofootshock was given, and the test minus training latency, to a ceiling of 180 s,called the retention score, was taken as a measure of memory retention(Jerusalinsky et al., 1993).

For the habituation training the rat was gently placed on the platform and leftto freely explore the box for 2 min. Step-down latency, numbers of step-downs


and step-ups, number of rearings and crossings of two marks in the grid werecounted. A signi®cant decrease in the number of crossings and rearings in the testsession was taken as a parameter of habituation (the di�erences were evaluated bythe two tailed, Mann±Whitney Ù' test) (see Wolfman et al., 1991).

After training, the rats were bilaterally microinjected with 0.5 ml of MTs in twodoses: 0.3 mg and 2 mg, or just the vehicle (0.1 M sodium phosphate bu�er insaline, pH 7.4) or the MTs plus the antagonist scopolamine (2 mg).

2.3.2. ResultsA control group was trained and tested without any additional treatment and

this was compared to the group injected with vehicle. As it has been previouslyshown, latency di�erences in both groups were not statistically signi®cant; themedian of which was about 40 s (24/63; interquartile range in seconds) and thedi�erences were evaluated by the two tailed Mann±Whitney Ù' test (see Izquierdoet al., 1992).

Table 1 is a summary of the results. MT2 (at the higher dose) showed similarfacilitatory e�ects to the muscarinic agonist oxotremorine when injected into thedorsal hippocampus of rats after an inhibitory avoidance task (median of latencydi�erence, 180 s), while a ®ve fold lower dose of MT2 still caused a signi®cantenhancement of test performance (median, 88 s). It was facilitatory and this e�ectwas blocked by scopolamine (Jerusalinsky et al., 1993). MT1 was facilitatory atthe lower dose but had no evident e�ects at higher doses. The facilitatory actionsof the peptides were no longer evident when the lower doses were given alongwith scopolamine.

MT3, in similar conditions, caused retrograde amnesia since there were nosigni®cant di�erencies in latencies of test session with respect to training session(Jerusalinsky et al., 1997b; 1998). This e�ect may be comparable to that ofscopolamine (Izquierdo et al., 1992; Jerusalinsky et al., 1993; 1997a).

Table 1

E�ect of intrahippocampal infusion of MT1, MT2 and MT3 after step-down inhibitory avoidance task

in the rat

Post-training injection into the hippocampus

MT1 MT2 MT3

Habituationa No e�ect Facilitatory No e�ect

Inhibitory avoidanceb Facilitatory Ð No e�ect Facilitatory Amnesic

a Habituation. Facilitatory: the decrease in number of crossings and rearings in the test session was

signi®cantly higher (P < 0.002) than in control group. No e�ect: there was no di�erence, compared to

control group (Mann±Whitney Ù' test, two tailed).b Inhibitory avoidance. Facilitatory: the retention scores increased in treated animals (i.e., median

180 s), compared to trained controls (median, 40 s), the score di�erences were signi®cant at P< 0.002

level. No e�ect: the di�erences between trained MTs injected and trained controls were not statistically

signi®cant. Amnesic: the scores were not signi®cantly di�erent to the training session and were signi®-

cantly lower than in trained controls (Mann±Whitney Ù' test, two tailed).


The e�ect of MT2 on the habituation task was also facilitatory, but MT1 wasnot e�ective at any dose. On the other hand, MT3 did not show any signi®cante�ect on habituation.

3. Discussion

3.1. Selectivity of muscarinic toxins for muscarinic receptor subtypes

We have shown that MT1 and MT2 selectively inhibit the binding of muscarinicantagonists to native and cloned MAChR. They mainly bind to m1 and m4cloned receptors (Jerusalinsky and Harvey, 1994; Kornisiuk et al., 1995b), whichare supposedly closely related to M1 and M4 pharmacologically characterizedreceptor subtypes.

The MTs we have studied did not bind to benzodiazepine-receptors, to gammaamino butyric acid receptors, to 125I-a-bungarotoxin sites (Jerusalinsky et al.,1994, 1995; Kornisiuk et al., 1995a), or to glutamate receptors of theaminohydroxymethylisoxazole propionic acid subtype (Kornisiuk, Jerusalinskyand Cammarotta, personal communication) and reversibly bound to a-adrenoceptors (Jerusalinsky et al., 1997c; Harvey et al., 1999).

Both MT1 and MT2 exhibited a rather low level of speci®c binding to atrialmembranes, rich in m2 receptors, (Jerusalinsky et al., 1994; Jolkkonen et al., 1995)but did not bind to pancreas membranes, rich in m3 receptors (Kornisiuk et al.,1995a; Harvey et al., 1999).

MT1 showed high a�nity for both m1 and m4 receptors (Fig. 1A), while MT2was rather selective for m1 (Fig. 1B) and MT3 was highly selective for m4 subtype(Fig. 1C). Potter and co-workers (1996) have shown that m4-toxin, same as MT3,bound to m1 receptor with 40 fold lower a�nity than to m4 (Ki at m1 subtype 78nM). Thus, our results corroborate their data, but we found lower a�nity to m1subtype, leading to a much wider gap between the a�nities for both receptors (Ki

1.1 mM at m1, compared to 1.38 nM at m4).From saturation curves performed in cloned MAChR with 3H±NMS, it had

been shown that the apparent dissociation constant Kd increased in the presenceof MT1 or MT2, while the concentration of binding sites was not markedlychanged (Kornisiuk et al., 1995b). These results suggested that the inhibition of3H±NMS binding was competitive, although another kind of interaction may notbe ruled out. It had also been shown that association curves of 3H±NMS tocloned receptors after preincubation with each one of the three MTs, did notreach the same plateau as in control association curves, suggesting that asubstantial portion of the toxin binding was irreversible or very slowly reversible.On the other hand, the binding to a-adrenoceptors was highly reversible(Jerusalinsky et al., 1997c; Harvey et al., 1999).

These results are in agreement with those previously obtained in rabbit vasdeferens, where a presynaptic putative M1 receptor (pharmacologically classi®ed)controls the release of the excitatory neurotransmitters ATP and noradrenaline by


a negative feed-back. The e�ect of both MT1 and MT2 was to reduce the twitcheselicited by nerve stimulation of the muscle in this isolated nerve-musclepreparation (Jerusalinsky and Harvey, 1994; Kornisiuk et al., 1995a). The decreasewas not reversed by washing (Harvey et al., 1999; Jerusalinsky et al., 1997b).Hence, it was proposed that MT1 and MT2 have agonist action at a m1presynaptic receptor in the rabbit vas deferens.

MT3 (m4-toxin) was proposed to be a muscarinic antagonist at m4 centralreceptors because it antagonizes the ACh inhibition of adenylyl cyclase stimulatedeither by forskoline or dopamine (Olianas et al., 1996, 1997, 1998). Similar assaysin rabbit vas deferens carried out in the presence of MT3, showed no evidente�ect of this toxin (Bradley et al., 1997).

3.2. Muscarinic toxins and localization of muscarinic receptors

The inhibition of the speci®c binding of the radiolabelled antagonists 3H±QNBor 3H±NMS to cerebral cortex synaptosomal membranes by either MT1 or MT2reached 50 to 60% (Adem et al., 1988; Jerusalinsky et al., 1989, 1992). On theother hand, it had been previously reported that inhibition of the speci®c bindingof 3H±QNB in similar experiments with brainstem membranes showed only 35%inhibition (Jerusalinsky et al., 1992). These results correlated with the relativeconcentration of the di�erent subtypes of MAChR. In the cerebral cortex, all thesubtypes are expressed, but m1 appears to be the most conspicuous receptor(CorteÂ s and Palacios, 1986; Frey and Howland, 1992); while in the brainstem,poor in MAChR, the most concentrated subtype appears to be m2 (Frey andHowland, 1992).

The hippocampal formation of the rat was suggested to have a higherproportion of m1 and m4 receptors (Wall et al., 1991; Yasuda et al., 1993). MT1and MT2 exhibited a similar behavior in total inhibition in synaptosomalmembranes prepared from this region (Kornisiuk et al., 1995a). About 50% of theMAChR in the same preparation from the hippocampus appeared insensitive tothe toxins (Kornisiuk et al., 1995a).

The striatum was reported as the region the most rich in m4 receptors (Boulayet al., 1996). In synaptosomal membranes, MT1 was much more e�ective ininhibiting the binding of 3H±NMS, reaching about 85% inhibition, with a Ki of84 nM, while with MT2 the inhibition was less than 50 % and the apparent Ki

was 1.1 mM (Kornisiuk et al., 1995a). These results allowed us to suggest that m1receptors would also be very well represented in the striatum.

Since MT1 was supposedly bound to both m1 and m4 receptors, it wasnecessary to clarify the relative proportion of these MAChR subtypes in thedi�erent regions of the brain. Adem and Karlsson (1997) had found that 125I-MT1preferentially binds to M1 receptor. We have selected MT2 and MT3 for theirde®ned selectivity at mainly one MAChR subtype (Fig. 1B and C). Theautoradiograms of brain slices labelled with 3H±NMS showed di�erent patterns ofinhibition with each one of the toxins (Fig. 2A and B). In this way we were ableto study the putative localization of m1 and m4 receptors in CA1, CA3 and the


dentate gyrus subregions of the hippocampal formation, as well as in frontalcortex and striatum (Fig. 2A and B). These structures subserve di�erent functions,being the hippocampal formation more involved in memory processing and thestriatum, more related to control of movement, although both are involved indi�erent learnings. On the other hand, the hippocampus and the frontal cortexalso appear to be involved with a�ection components of learning and memory.

As a general observation, the levels of m1 and m4 receptors determined byusing MT2 and MT3 are in agreement with those reported by other methods inthe cerebral cortex (CorteÂ s and Palacios, 1986; Frey and Howland, 1992; Ademand Karlsson, 1997), in the hippocampus (Wall et al., 1991; Yasuda et al., 1993;Adem and Karlsson 1997) and in the striatum (Boulay et al., 1996). However,there were some signi®cant di�erences that have revealed once again thedi�culties in discriminating MAChR subtypes in situ.

There appear to be similar proportions of m1 and m4 subtypes in the cerebralcortex and their concentrations are lower than in the hippocampal formation,except in the CA3 region where they exhibit concentration levels similar or slightlylower than those in the cortex, in agreement with results of Adem and Karlsson(1997) for m1 receptors. It was not possible to reveal such di�erences insynaptosomal membranes where all the regions of the hippocampus were mixedup (Kornisiuk et al., 1995a).

Then, among the quanti®ed regions, the highest level of m1 receptors would belocated in the dentate gyrus of the hippocampal formation and not in the cerebralcortex (Fig. 2A), and most of the remaining muscarinic receptors of the dentategyrus would belong to the m4 subtype (Fig. 2B). There is also a high proportionof m1 in the CA1 region, being the m4 subtype is also very conspicuous in CA1(Fig. 2A and B). Hence, although m1 and m4 MAChR are very well representedin the hippocampus, there are clear regional di�erences.

Most of the receptors in the striatum appeared to be m4 (more 70%) and someof the remaining MAChR seem to belong to the m1 subtype (Fig. 2A and B).This is in agreement with the data from the literature, although there is also asmall proportion of m2 receptors there (Levey, 1993; Rose et al., 1998). Takinginto account the 125I-MT1 binding sites, it has been reported that there is a higherproportion of m1 receptors in the striatum than in the other regions (Adem andKarlsson, 1997).

It should be noticed that the striatum has been considered the most richstructure in m4 subtype. However, our data suggest that m4 receptors arerepresented only at a slightly lower concentration in the dentate gyrus (i.e., 540compared to 430 fmol/mg tissue).

3.3. Muscarinic toxins and memory

As muscarinic receptors have been considered to be involved in memoryconsolidation (see Izquierdo, 1989; Izquierdo et al., 1992; Jerusalinsky et al.,1997a) we performed assays injecting either the muscarinic antagonist scopolamineor the agonist oxotremorine into the dorsal hippocampus, immediately after an


inhibitory avoidance task. The retention test was performed 24 h later. The ratlearns to avoid a foot shock by remaining on an isolated platform. The animalthat received scopolamine showed retrograde amnesia, while the one withoxotremorine improved its performance (Izquierdo et al., 1992).

Thus, we decided to use the MTs to ®nd out which receptor subtype might beinvolved in this mechanism of memory consolidation. In 1993 we showed an invivo e�ect of the toxins for the ®rst time (Jerusalinsky et al., 1993). The rats weretrained in an inhibitory avoidance task. Immediately afterwards they received aninjection of MT1 or MT2 into the dorsal hippocampus, or one of the toxins plusscopolamine. MT2 improved retention of the task and its e�ect was antagonizedby scopolamine, since the joint infusion produced either a decrease of performanceor amnesia, depending on the dose. Similar e�ects were found with MT2 in thehabituation task. MT1 showed a similar e�ect in the retention of the inhibitoryavoidance task at the lower doses (Jerusalinsky et al., 1994, 1995) while it had noe�ect at higher doses at which MT2 still showed a facilitatory action (Table 1).MT1 did not show any evident e�ect on habituation. Taking into account thatMT2 toxin was highly selective for m1 receptors and that there is a highconcentration of these receptors in the hippocampal formation, that this toxinbehaved as an agonist at m1 receptor of the rabbit vas deferens, and that thee�ect on memory retention of this task resembled the action of the muscarinicagonist oxotremorine and was reversed by the antagonist scopolamine, weproposed that the m1 receptor of the dorsal hippocampus is directly involved inpositive modulation of memory of this task (Jerusalinsky et al., 1993). We do notknow which action MT1 and MT2 have at m4 receptors. They may be eitheragonists or antagonists. Further investigations are needed to clarify thepharmacological pro®le.

MT3 injected into the same region caused amnesia (Table 1). (Jerusalinsky etal., 1998) Since this toxin is highly selective for m4 receptor (Fig. 1) we suggestthat m4 receptor subtype in the dorsal hippocampus is involved, at least inmodulation, in memory consolidation of a step-down inhibitory avoidance, a taskwith spatial and strong aversive components. Since MT3 has been shown to be anantagonist at central muscarinic receptors (Olianas et al., 1996, 1997), our resultsalso suggest a positive modulation of memory consolidation by hippocampal m4receptors. However, this receptor subtype did not seem to be involved in a morespatial-olfactory (exploratory) task as the habituation to a novel environment, atleast in the conditions of these experiments. A putative explanation for the lack ofe�ect of higher doses of MT1 in the retention of the inhibitory avoidance may bethat MT1, a toxin with high a�nity for m1 and m4 receptors, would behave as anantagonist at the m4 subtype. However, this requires further investigation.

Acknowledgements

We are grateful for the ®nancial support of the International Foundation forScience, the University of Buenos Aires and the National Council of Scienti®c and


Technological Investigations. We acknowledge the collaboration of Professor AlanHarvey and his group at Strathclyde University in functional studies.

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Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system

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