gerard j. marek et al- physiological antagonism between 5-hydroxytryptamine2a and group ii...

8/3/2019 Gerard J. Marek et al- Physiological Antagonism between 5-Hydroxytryptamine2A and Group II Metabotropic Glutam

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Physiological Antagonism between 5-Hydroxytryptamine2A andGroup II Metabotropic Glutamate Receptors in PrefrontalCortex1

GERARD J. MAREK, REBECCA A. WRIGHT, DARRYLE D. SCHOEPP, JAMES A. MONN, and GEORGE K. AGHAJANIAN

Departments of Psychiatry and Pharmacology, Yale University School of Medicine, Ribicoff Research Facilities of the Connecticut MentalHealth Center, New Haven, Connecticut (G.J.M., G.K.A.); Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (R.A.W.,D.D.S., J.A.M.).

Accepted for publication September 20, 1999 This paper is available online at http://www.jpet.org

ABSTRACT

In prefrontal cortex, 5-hydroxytryptamine2A (5-HT2A) receptorshave been linked to the action of hallucinogens and atypicalantidepressant/antipsychotic drugs. Previously, we haveshown in cortical layer V pyramidal cells that a nonselectivemetabotropic glutamate (mGlu) receptor agonist suppressesthe induction of excitatory postsynaptic potentials/currents(EPSPs/EPSCs) via activation of 5-HT2A receptors. In thisstudy, we tested the ability of the selective mGlu2/3 agonist(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylatemonohydrate (LY354740) and the selective mGlu2/3 antagonist2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl-

)propanoic acid (LY341495) to modulate serotonin(5-HT)-in-duced EPSPs and electrically evoked EPSPs by using intracel-lular recording from layer V pyramidal cells in medial prefrontalcortex. The mGlu2/3 antagonist LY341495 increased the fre-quency and amplitude of 5-HT-induced EPSCs, suggesting arole for mGlu2/3 receptors in mediating the action of endoge-

nous glutamate on autoreceptors. Conversely, the mGlu2/3agonist LY354740 was highly effective and potent (EC50 89nM) in suppressing glutamate release induced by 5-HT2A re-ceptor activation in the medial prefrontal cortex, probably via apresynaptic mechanism. The mGlu2/3 antagonist LY341495potently blocked the suppressant effect of LY354740 on 5-HT-induced EPSCs as well as electrically evoked early EPSPs.

Autoradiography with the radioligands [3H]LY354740 and[125I]()-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane showsa striking overlap of the laminar distribution of mGlu2/3 and5-HT2A receptors in the medial prefrontal cortex that is notapparent in other cortical regions. These findings suggest aclose coupling between mGlu2/3 and 5-HT2A receptors in theprefrontal cortex that may be relevant for novel therapeuticapproaches in the treatment of neuropsychiatric syndromessuch as depression and schizophrenia.

5-Hydroxytryptamine2A (5-HT2A) receptor antagonistsblock the psychotomimetic effects of hallucinogens in hu-mans (Vollenweider et al., 1998) and are thought to contrib-ute to the therapeutic effects of atypical antidepressant/an-tipsychotic drugs (Kroeze and Roth, 1998; Marek and

Aghajanian, 1998b). Furthermore, increased glutamate re-lease in the prefrontal cortex appears to be a common feature

shared by both noncompetitive N-methyl-D-aspartate antag-onists and hallucinogenic drugs (Aghajanian and Marek,1999b), both of which mimic some of the symptoms of acutepsychosis. 5-HT2A receptor activation increases the fre-quency of spontaneous (nonelectrically evoked) excitatorypostsynaptic currents (EPSCs) in apical dendrites of neocor-tical layer V pyramidal cells in a novel manner, suggestingfocal release of glutamate from discrete pathways (Aghaja-

nian and Marek, 1997). This induction of an increase in thefrequency of EPSCs by serotonin (5-HT) is completelyblocked by the -amino-3-hydroxy-5-methylisoxazole-4-pro-pionate (AMPA)/kainate antagonist LY293558 (Aghajanianand Marek, 1997). The ability of 5-HT2 antagonists to block5-HT-induced EPSCs is clearly mediated through 5-HT2A,

Received for publication July 9, 1999.1 This work was supported by United States Public Health Service Grants

K08 Award (National Institute of Mental Health; G.J.M.) and MH17871 (Na-tional Institute of Mental Health; G.K.A.), a National Alliance for Research onSchizophrenia and Depression (NARSAD) 1999 Fairfax Investigator Award(G.J.M.), the State of Connecticut (G.J.M., G.K.A.), and Lilly Research Labo-ratories, Eli Lilly and Company, Indianapolis, IN.

ABBREVIATIONS: 5-HT2A, 5-hydroxytryptamine2A; mGlu, metabotropic glutamate; EPSPs, excitatory postsynaptic potentials; EPSCs, excitatory

postsynaptic currents; 5-HT, serotonin; L-SOP, L-serine-O-phosphate; LY354740, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate

monohydrate; LY379268, ( )-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate; LY341495, (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-

yl)-3(xanthy-9-yl)propanoic acid; LY366563, (1R,2R,5S,6R)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate; DOI, ()-1-(2,5-dime-

thoxy-4-iodophenyl)-2-aminopropane; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionate; TTX, tetrodotoxin; ACSF, artificial cerebro-

spinal fluid.

0022-3565/00/2921-0076$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 292, No. 1Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.JPET 292:7687, 2000

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rather than 5-HT2C, receptors (Aghajanian and Marek, 1997;Marek and Aghajanian, 1999).

The potent partial 5-HT2A/2C agonist ()-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a hallucinogenic drug,induces an increase in the frequency of EPSCs, reaching only10% of the level for 5-HT itself. However, DOI induces anincrease in the late component of electrically evoked EPSCs,an effect which is blocked by the selective 5-HT2A antagonistand putative antipsychotic drug M100,907 (Aghajanian andMarek, 1999a). We have previously suggested that a commonmechanism of glutamate release may underlie both the in-creased frequency of 5-HT-induced EPSCs and electricallyevoked late EPSPs occurring after DOI application (Aghaja-nian and Marek, 1999a). This common mechanism may in-

volve the asynchronous pathway of glutamate release be-cause addition of Sr2 to a Ca2-free artificial cerebrospinalfluid (ACSF) supported 5-HT-induced EPSCs under condi-tions in which the synchronous pathway of glutamate re-lease was blocked (Goda and Stevens, 1994; Aghajanian andMarek, 1999a). Furthermore, addition of Sr2 to a Ca2-free

ACSF also supported electrically evoked late EPSCs underconditions in which the evoked early EPSCs, via the synchro-

nous pathway of glutamate release (Goda and Stevens,1994), were blocked. The electrically evoked late EPSCs afterDOI application appeared similar to the electrically evokedlate EPSCs from the Sr2 substitution experiments. Whetherthe electrically evoked late EPSCs represent asynchronousrelease of transmitter or conventional polysynaptic EPSCs,agents that suppress prefrontal glutamate release inducedby activation of 5-HT2A receptors could provide a novel ther-apeutic approach to the treatment of depression and schizo-phrenia (Marek and Aghajanian, 1998b).

One possible approach to suppressing glutamate release isthrough metabotropic glutamate (mGlu) receptors becausemany subtypes function as presynaptic autoreceptors on glu-tamatergic terminals (Conn and Pin, 1997). The mGlu recep-

tors are a novel family of glutamate G-protein coupled recep-tors that are commonly separated into three classes based onboth pharmacology and signal transduction pathways (Connand Pin, 1997; Schoepp et al., 1999). Group I mGlu receptors(e.g., mGlu1 and mGlu5) are coupled to phospholipase C andphosphoinositide hydrolysis. In contrast, both group II (e.g.,mGlu2 and mGlu3) and group III (e.g., mGlu4, mGlu6,mGlu7, and mGlu8) are negatively coupled to cAMP forma-tion and are thought to function as inhibitory presynapticautoreceptors that may play a role in synaptic plasticity(Conn and Pin, 1997; Li et al., 1998). These group II andgroup III mGlu receptors have overlapping, but distinct, pat-terns of mRNA expression in the rat central nervous system(Ohishi et al., 1993a,b, 1995; Saugstad et al., 1997).

Previously, a relatively nonspecific group II/III mGlu ago-nist, (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid(ACPD; 200 M), was found to suppress 5-HT-induced EP-SCs (Aghajanian and Marek, 1997). Recently, novel conforma-tionally constrained analogs of glutamate that are selective forthe group II mGlu receptors at low concentrations, includingtwo agonists, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and ()-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), andan antagonist, (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495), have been devel-oped (Monn et al., 1997, 1999; Schoepp et al., 1997; Fitzjohn

et al., 1998; Kingston et al., 1998). We now provide evidencethat physiological as well as pharmacological activation ofmGlu2/3 receptors suppresses glutamate release induced by5-HT2A receptor activation in the medial prefrontal cortex(transitional neocortex including both the prelimbic area ofthe medial prefrontal cortex and the anterior cingulate). Fur-thermore, a striking laminar overlap was found in the medialprefrontal cortex compared with other cortical regions for5-HT2A and mGlu2/3 receptor binding that may be relevantto targeting drugs for neuropsychiatric syndromes involvingthe prefrontal cortex.

Materials and Methods

Autoradiography. Whole brains were obtained from adult maleSprague-Dawley rats (120200 g), rapidly frozen in powdered dryice, and mounted on cryostat chucks. Coronal sections (20 m) werecut from the prefrontal cortex and thaw-mounted on gelatin-coatedslides. Sections were stored at 20C before use. For [3H]LY354740binding, tissue sections were preincubated in ice-cold 10 mM potas-sium phosphate buffer with 100 mM potassium bromide (phosphate/bromide buffer), pH 7.6, for 30 min to remove endogenous receptorligands, then rapidly dried under a stream of cool air. Then the

sections were incubated for 60 min at room temperature in phos-phate/bromide buffer with 50 nM [3H]LY354740 (custom prepared by Amersham, Buckinghamshire, England). Nonspecific binding wasdetermined using adjacent sections with 1 mM L-glutamate in thebuffer solution. After incubation, each slide was rinsed rapidly threetimes with 2 ml of ice-cold phosphate/bromide buffer followed by afinal rinse with 2 ml of ice-cold glutaraldehyde (49%)/acetone mix-ture (1:19, v/v), then quickly dried under a stream of warm air. Rinsetime was 10 s per slide. Sections were opposed, with tritium mi-croscales, to tritium-sensitive film (Hyperfilm-3H, Amersham, Pisca-taway, NJ) for 14 days.

For measurement of 5HT2A binding sites, tissue sections from thesame rats were incubated for 90 min at room temperature in a pH 7.4buffer (50 mM Tris/HCl, 4 mM CaCl2, 0.1% ascorbate, and 0.1%bovine serum albumin) with 0.5 nM [125I]DOI (NEN Life Science

Products, Inc., Boston, MA). Nonspecific binding was defined byincubation of adjacent sections in buffer and radioligand with 1 MM100,907 (formerly known as MDL100,907) added. After incubation,the slides were rinsed twice for 10 min in ice-cold assay bufferfollowed by a brief immersion in ice-cold purified water (Milli-Q;Millipore, Bedford, MA), then dried under a stream of warm air.Tissue sections were opposed, with 125I microscales, to 125I-sensitivefilm (Amersham, Piscataway, NJ) for 30 h.

Quantitative autoradiogram analysis was performed with a com-puter-assisted image analyzer (MCID; Imaging Research Inc., St.Catherines, Ontario). Optical density values were converted to fem-tomole per milligram of protein by using a computer generatedregression analysis. Values displayed in Fig. 4 represent data aver-aged from four rats with six sections per rat. This data was analyzedwith a one-way ANOVA and testing of layers significantly different

from layer Va with the Neumann-Keuls multiple comparison test(GraphPad Prism; GraphPad Software, Inc., San Diego, CA).Membrane Binding Methods. Well washed crude membranes

of the rat forebrain were prepared as described previously (Wright etal., 1994). Cell membranes from human mGlu2- and human mGlu3-expressing RGT cells (cells transfected with a rat glutamate trans-porter) were obtained as described previously (Johnson et al., 1999).Frozen tissue pellets were thawed on the day of assay and washedthree times with ice-cold assay buffer (phosphate/bromide buffer). Tostart the reaction, rat tissue (0.150.20 mg of protein) or mGlu2 ormGlu3 tissue (0.04 0.06 mg of protein) was added to deep-wellpolypropylene microtiter plates or 5-ml scintillation vials (for ratbrain tissue) that contained [3H]LY354740 (10 nM) and appropriateconcentrations of test compounds in assay buffer. Final assay volume

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was 0.5 ml. Nonspecific binding was defined with 1 mM L-glutamate.Samples were incubated on ice for 60 min and bound radioligand wasseparated from free radioligand by rapid filtration with washes with1 ml of ice-cold assay buffer using a Whatman GF-B filter for mGlu2or mGlu3 binding. For rat brain tissue, bound [3H]LY354740 wasseparated from free [3H]LY354740 by centrifugation as describedpreviously (Wright et al., 1994). Protein concentration was deter-mined by using the Pierce Coomassie micro assay (Rockford, IL). Kivalues were determined by using nonlinear regression in the Graph-

Pad Prism program (GraphPad Software, Inc.).Electrophysiology. Brain slices were prepared from maleSprague-Dawley rats (120200 g) as described previously (Aghaja-nian and Rasmussen, 1989). Briefly, rats were anesthetized withchloral hydrate (400 mg/kg, i.p.) and decapitated. Coronal slices (500m) were cut with an oscillating-blade tissue slicer at a level corre-sponding to approximately 2.5 mm anterior to bregma (Paxinos andWatson, 1986). A slice containing the medial prefrontal cortex wasthen transferred to the stage of a fluid-gas interface chamber, whichhad a constant flow of humidified 95% O2, 5% CO2. The slices wereperfused in a chamber heated to 34C with normal ACSF, whichconsisted of 126 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 26mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM D-glucose.

Intracellular recording and single-electrode voltage clamping wereconducted in layer V pyramidal cells by using an Axoclamp-2A (Axon

Instruments, Inc., Foster City, CA) as previously described (Aghaja-nian and Marek, 1997). Stubby electrodes (8 mm, shank to tip)with relatively low capacitance and resistance (3060 M) werefilled with 1 M potassium acetate. The cells were voltage clamped at70 mV. The EPSCs recorded under these conditions do not appearto be contaminated by reversed inhibitory postsynaptic currents forthe following reasons. Only a small fraction of 5-HT-induced EPSCs(15%) are blocked by bicuculline during intracellular recordingsusing KCl-containing electrodes, suggesting the presence of somereverse inhibitory postsynaptic currents. In contrast, the 5-HT-in-duced EPSCs recorded with nonchloride-containing electrodes (i.e.,potassium acetate or gluconate) at holding potentials near ECl arecompletely blocked by the AMPA/kainate antagonist LY293558(Aghajanian and Marek, 1997). The voltage-clamp signals were low-pass filtered (1000 Hz) and data were acquired with a pCLAMP/

Digidata 1200 system (Axon Instruments, Inc.). EPSC frequencieswere obtained from 10 successive episodes (1-s duration) during thebaseline and drug treatment periods. Evoked potentials were ob-tained while holding cells at 80 mV and stimulating the forcepsminor in the white matter deep in the cortex.

EPSC frequency and amplitude were determined with Mini Anal-ysis Program (www.synaptosoft.com; Synaptosoft, Inc., Leonia, NJ)using thresholds of 10 pA and an area of150 fCs1 for synapticcurrents. Statistical comparisons of within-cell responses were madewith two-tailed paired t-tests requiring P .05 for statistical signif-icance. The Kolmogorov-Smirnov test was used to determinewhether significant changes occurred in the EPSC amplitude cumu-lative probability plots.

The determination of EC50 values for the suppression of 5-HT-induced increases in EPSC frequency or of evoked EPSPs werecalculated by nonlinear curve fitting (DeltaGraph 4.0; DeltaPoint,Monterey, CA). The Schild equation was used to calculate pA2 values

for the mGlu antagonist LY341495 to effect a shift in the concentra-tion-response curve for LY354740 (Arunlakshana and Schild, 1959).

Compounds. The drugs used in this study were obtained from thefollowing suppliers: Sigma Chemical Co. (St. Louis, MO; 5-HT crea-tine sulfate), Research Biochemicals (Natick, MA; AMPA), and Alomone Labs [Jerusalem, Israel; tetrodotoxin (TTX)]. LY354740,(1R,2R,5S,6R)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate mono-hydrate (LY366563), LY379268, and LY341495 were prepared byJ.A.M. at the Lilly Research Laboratories (Indianapolis, IN).

Results

Binding Affinity of mGlu2/3 Agonists and Antago-

nist. The mGlu2/3 agonist LY354740 potently bound to hu-man mGlu2 and mGlu3 receptors with a slight selectivity formGlu2 compared with mGlu3 (Table 1; Fig. 1), consistentwith a previous report of a5-fold selectivity of LY354740 formGlu2 versus mGlu3 in a functional assay (Schoepp et al.,1997). LY379268, another mGlu2/3 agonist, potently boundto human mGlu2 and mGlu3 receptors with a slight selectiv-ity for mGlu2 versus mGlu3 (Table 1; Fig. 1), but selectivityfor LY379268 at mGlu2 versus mGlu3 receptors has not beenobserved in a functional assay or in a binding assay (Monn et

al., 1999) using the antagonist LY341495 as the radioligand(Johnson et al., 1999). LY341495 bound with a nanomolarpotency to both mGlu2 and mGlu3, but with 3-fold selec-tivity for mGlu3 versus mGlu2. All three mGlu2/3 ligandsfully displaced [3H]LY354740 from rat forebrain bindingsites with nanomolar potency (Fig. 1). Importantly, the rankorder of potency for all three mGlu2/3 ligands at displacingthe radioligand from the rat forebrain binding sites wassimilar to that found with human mGlu2/3 receptors trans-fected into the RGT cells.

Electrophysiological Characteristics of Cortical

Layer V Pyramidal Cells. Layer V pyramidal cells wererecorded in a zone ca. 1/2 to 2/3 the distance between the pialsurface and the subcortical white matter. The pyramidal cells

in the present study had the following characteristics: restingpotential, 70.7 0.8 mV; action potential amplitude,81.0 0.8 mV; action potential duration (at half-amplitude),0.78 0.02 ms; input resistance (0.4 nA test pulse), 34.3 2.3 M (n 71). All of the cells in the present series, excepta single cell with an intrinsically bursting firing pattern to aconstant depolarizing pulse, had the previously reportedcharacteristics (McCormick et al., 1985; Connors and Gut-nick, 1990) of regularly spiking pyramidal cells.

The mGlu2/3 Antagonist LY341495 Enhances the

Frequency and Amplitude of 5-HT-Induced EPSCs. Wehad previously observed that the AMPA/kainate antagonistLY293558 and the nonselective mGlu agonist (1S,3S)-1-ami-nocyclopentane-1,3-dicarboxylic acid (ACPD) completelyblocked 5-HT-induced EPSCs in a fashion suggesting that

TABLE 1

Affinities for displacement by LY354740, LY379268 and LY341495 at cloned human mGlu receptors (mGlu2 and mGlu3) and rat brainhomogenates of [3H]LY354740 binding. All values displayed are means S.E. (n 3).

CompoundhmGlu2 hmGlu3 Rat forebrain

KI 95% CI KI 95% CI KI 95% CI

nM nM nM

LY354740 47.8 15 24.764.4 65.8 27 66.2146 21.4 2.0 17.227.5LY379268 6.65 1.3 4.039.08 12.8 5.3 5.7316.9 1.45 0.18 0.852.08LY341495 6.01 1.3 3.409.96 2.52 0.6 1.393.97 0.68 0.1 0.311.08

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these drugs may be acting at a postsynaptic and a presynap-tic site, respectively (Aghajanian and Marek, 1997). To testwhether tonic release of endogenous glutamate might beacting on inhibitory autoreceptors to reduce the sustainedincrease in EPSCs induced by 5-HT, the effects of the highly

selective mGlu2/3 antagonist LY341495 (Kingston et al.,1998) on 5-HT-induced EPSCs were investigated with intra-cellular recordings from layer V pyramidal cells of the medialprefrontal cortex. It should be noted that LY341495 (1 M)does not block the suppressant action of the group III mGlureceptor agonist L-serine-O-phosphate (L-SOP) on 5-HT-in-duced EPSPs (unpublished observations, G.J.M.). ThemGlu2/3 antagonist LY341495 (20 min) enhanced the fre-quency of 5-HT-induced EPSCs by 30 to 65% (0.130 M; Fig.2; Table 2); a significant increase in the frequency of EPSCswas observed first at 100 nM, although a trend was presentfor a significant increase at 30 nM (P .09, n 4). Theclassical interpretation of a change in the frequency of syn-aptic currents is via an effect on the presynaptic terminal(Van der Kloot, 1991). LY341495 also increased the ampli-tude of 5-HT-induced EPSCs by 12 to 21% (100 nM to 30 M;Fig. 2; Table 1). In 16 of the 19 cells (4 cells were tested withtwo or more concentrations), LY341495 significantly in-creased the EPSC amplitude for the 5-HT-induced EPSCs(Kolmogorov-Smirnov test, P .05 to P .0001, Fig. 2B). Achange in the amplitude of synaptic currents could be medi-ated by either a presynaptic or postsynaptic effect (Van der

Kloot, 1991). LY341495 did not alter the amplitude of elec-trically evoked early EPSPs (data not shown, n 2) or theamplitude of spontaneously occurring EPSCs (contrast Fig.2, A1 and A3; Table 2) observed under basal conditions in 17of the 19 cells tested (Kolmogorov-Smirnov test, P .05).Together, these results suggest that mGlu2 and/or mGlu3function as autoreceptors that are tonically activated byphysiological concentrations of glutamate released via5-HT2A receptor activation.

mGlu 2/3 Agonists Suppress 5-HT-Induced EPSCs

and Electrically Evoked EPSPs. Intracellular recordingsfrom layer V pyramidal cells of the medial prefrontal cortexwere used to test mGlu2/3 agonists for both the suppressionof 5-HT-induced EPSCs and the increase in electrically

evoked late EPSPs after application of the 5-HT2A agonistDOI. LY354740 (Schoepp et al., 1997) was virtually equipo-tent at suppressing 5-HT-induced EPSCs (n 5) and DOI-enhanced electrically evoked late EPSPs (n 5) (EC50 val-ues: 89.1 versus 85.3 nM). In contrast, LY354740 was 3- to5-fold less potent at suppressing electrically evoked earlyEPSPs (EC50 values: 235 and 454 nM; Table 3; Fig. 3C; Fig.4, bottom). LY366563 (Monn et al., 1997; Schoepp et al.,1998) (1 M), the inactive enantiomer of LY354740, did notsuppress 5-HT-induced EPSCs in any of the four cells fromthe medial prefrontal cortex tested (data not shown). Thesuppressant effect of LY354740 on 5-HT-induced EPSCsshowed regional variability in that neurons from the medialprefrontal cortex were 4-fold more sensitive to this drug thanwere a random sample of cells (n 3) from the Fr1, Fr3, andPar1 areas of the frontoparietal neocortex (Paxinos andWatson, 1986) (EC50 398 142, n 3). Another mGlu2/3agonist, LY379268 (Table 3), was approximately equipotentin suppressing 5-HT-induced EPSCs and the electricallyevoked early EPSPs and late EPSPs after DOI application(EC50 values: 230276 nM, n 4).

A Selective mGlu2/3 Antagonist Blocks the Effects of

LY354740.A concentration of mGlu2/3 antagonist LY341495that does not appear to block the suppressant action by themGlu4/6/7/8 receptor agonist L-SOP on 5-HT-induced EPSPs(1 M, G.J.M., unpublished observations) was tested

Fig. 1. Displacement of [3H]LY354740 binding by mGlu2/3 ligands. Eachpanel shows the displacement of [3H]LY35740 binding by the mGlu2/3agonists LY354740 and LY379268, and the mGlu2/3 antagonistLY341495 in cells transfected with either human mGlu2 receptors (top),human mGlu3 receptors (middle), or to rat forebrain binding sites (bot-tom).



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against the suppressant action of the mGlu2/3 agonistLY354740. LY341495 resulted in a robust rightward shift inthe concentration-response curve for the suppression byLY354740 against the frequency of 5-HT-induced EPSCs,yielding a pA2 value in the nanomolar range (pA2 7.27, n5, Fig. 4). LY341495, which is 3-fold more potent at mGlu3than mGlu2 receptors, was 4-fold more potent at blockingthe suppressant effect of LY354740 on electrically evokedearly EPSPs (pA2 7.9, n 5, Fig. 4) than on 5-HT-inducedEPSCs (Table 1). The antagonism by LY341495 against sup-pression by LY354740 for both the 5-HT-induced EPSCs andthe electrically evoked early EPSPs was surmountable byhigher concentrations of LY354740, which is consistent withcompetitive antagonism. The potency of LY341495 in block-ing the suppressant action of LY354740 on electricallyevoked late EPSPs after DOI application was not tested.

Presynaptic versus Postsynaptic Action of

LY354740. The 5-HT-induced EPSCs are mediated viaAMPA/kainate receptors on layer V pyramidal cells becausethey are completely blocked by the AMPA/kainate antagonistLY293558 (Aghajanian and Marek, 1997). To evaluatewhether a postsynaptic action could be involved in the sup-pression by mGlu agonists of the 5-HT-induced EPSCs, theeffect of LY354740 on inward currents induced by bath ap-plication of AMPA (5 M) was examined in layer V pyramidalcells. After treatment of the slice with TTX (2 M) to blockimpulse flow, LY354740 did not block AMPA-induced inwardcurrents (AMPA, 243 74 pA; LY354740 and AMPA, 233 75 pA; n 4; Fig. 5). LY354740 did induce small outwardcurrents in two of the four cells after TTX treatment (40 and60 pA). The effects of LY354740 and AMPA after TTX treat-ment were similar when the experiment was repeated by

Fig. 2. A mGlu2/3 antagonistenhances 5-HT-induced synap-

tic currents. A, in this layer Vpyramidal cell, the mean basalEPSC frequency and amplitudewere 2.5 s1 and 57.0 pA (A1).The mean 5-HT-induced EPSCfrequency and amplitude were31.3 s1 and 72.7 pA (A2). Themean frequency and amplitudeof EPSCs after a 20 min applica-tion of LY341495 (1 M) were1.8 s1 and 57.0 pA (A3). Note in

A3 that LY341495 does not alterthe frequency or amplitude ofEPSCs from basal (A1) condi-tions. After LY341495 applica-tion in this cell, the 5-HT-in-duced EPSC frequency and

amplitude was 61.1 s

1

and 99.6pA (A4). All tracings were takenfrom the same cell, in which thecommand potential was set at70 mV. B, an EPSC amplitudecumulative frequency plot in asingle cell for the effect of 5-HTboth before and after a 20 mintreatment with LY341495. C, asummary of experiments (n 46) from 30 nM to 30 MLY341495. The mean 5-HT-in-duced EPSC frequencies andamplitudes (mean S.E.) forthe basal, 5-HT, LY354740, andLY354740/5-HT conditions aredisplayed in Table 1. , signifi-cantly different from 5-HT

alone, P .05. , significantlydifferent from 5-HT alone, P .01.



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applying the drugs in ACSF containing no Ca2 (n 2, datanot shown).

A more direct test of whether the suppressant effect ofLY354740 on 5-HT-induced EPSCs was mediated via a pre-synaptic site involved analyzing the cumulative probabilitydistributions of the EPSC amplitudes. In four of five cells, a

concentration of LY354740 at approximately the EC50 forreducing the frequency of 5-HT-induced EPSCs did not causea significant change (P .05) in the EPSC amplitude asassessed with the Kolmogorov-Smirnov test (Fig. 5B). Theselective blockade in the frequency rather than the ampli-tude of 5-HT-induced EPSCs also argues for a presynapticlocus of action for LY354740 (Van der Kloot, 1991).

[125I]DOI and [3H]LY354740 Autoradiography. To be-gin an examination of the neuroanatomical basis of the in-teraction between 5-HT2A receptors and the group II mGlu(mGlu2 and mGlu3) receptors, we compared the autoradio-graphic pattern of binding for [125I]DOI and [3H]LY354740(Schoepp et al., 1997) in coronal brain sections including themedial prefrontal cortex by using concentrations of radioac-

tive and displacement ligands to specifically label 5-HT2Aand mGlu2/3 receptors, respectively (Figs. 6 and 7). Both[125I]DOI and [3H]LY354740 bound to the superficial andmid-layer of the medial prefrontal cortex. The laminar dis-tribution of [125I]DOI is consistent with previous observa-tions for peaks in specific 5-HT2A receptor binding in layers Iand Va of the neocortex and transitional cortex (Blue et al.,1988). In all areas of the neocortex and transitional neocor-tex, the [3H]DOI binding was highest in layer Va and signif-icantly lower in the intermediate layers (layers IIIV). Incontrast to the medial prefrontal cortex where [3H]LY354740binding was significantly lower in layers II/III than in layer

Va, in the frontoparietal region (e.g., Par1), [3H]LY354740binding was significantly higher in layers II/III/IV than inlayer Va.

Discussion

The most striking finding from the present studies wasthat, in the absence of an exogenous agonist, the potentgroup II mGlu antagonist LY341495 increased the frequencyand amplitude of 5-HT-induced EPSCs. This increase oc-curred at concentrations of LY341495 (1 M) that do notblock the suppressant action on 5-HT-induced EPSCs by thegroup III mGlu agonist L-SOP. The mGlu antagonist did notalter the amplitude of either the basal EPSCs or the electri-

cally evoked early EPSPs, these being situations where notonic activation of presynaptic autoreceptors by endogenousglutamate would be expected. However, when a vigorous andsustained increase in EPSCs occurred during the applicationof 5-HT, the frequency and amplitude of synaptic currentswas almost always enhanced by the mGlu antagonist. These

findings are consistent with the hypothesis that mGlu2/3receptors function as inhibitory autoreceptors in cortical glu-tamatergic terminals whose transmitter release is positivelyregulated by 5-HT2A receptor activation. These observationsappear to be similar to the previous demonstration in thehippocampus of a use-dependent activation of presynapticmGlu receptors that function to decrease excitatory aminoacid release (Scanziani et al., 1997).

Conversely, mGlu2/3 agonists suppress glutamate releaseinduced by 5-HT2A receptor activation from nerve endingsthat terminate onto cortical layer V pyramidal cells. Twopotent and selective mGlu2/3 agonists, LY354740 (Monn etal., 1997; Schoepp et al., 1997) and LY379268 (Monn et al.,1999), suppressed the increase in EPSCs induced by 5-HT

and the enhancement of electrically evoked late EPSPs in-duced by the partial 5-HT2A agonist DOI with a similarpotency. This action was not shared by LY366553, the inac-tive ()-isomer of LY354740. Furthermore, the selectivemGlu2/3 antagonist LY341495 blocked the suppressant ac-tion of LY354740 on 5-HT-induced EPSCs or electricallyevoked early EPSPs with potencies consistent with pharma-cological antagonism of mGlu2/3 receptors (Kingston et al.,1998).

Although both mGlu2/3 agonists LY354740 and LY379268suppressed 5-HT-induced EPSCs, the 3-fold greater potencyof LY354740 over LY379268 is surprising in light of thegreater affinity of LY379268 over LY354740 at mGlu2,mGlu3, and rat forebrain binding sites. In this context, itshould be noted that LY354740 and LY379268 are conforma-tionally constrained analogues of glutamic acid (Monn et al.,1997, 1999). Perhaps differences in the potency of these ago-nists in receptor binding assays compared with functionalassays in tissue slices could reflect differences in the abilityof these compounds to access the receptor due to differingaffinities at other synaptic sites such as glutamate transport-ers.

The mGlu2/3 agonists appear to act on a presynaptic site todecrease glutamate release. In four of five cells, LY354740did not alter the amplitude of 5-HT-induced spontaneousEPSCs at a concentration that decreased frequency by 50%.

TABLE 2

Effect of 5 HT and the mGlu2/3 antagonist LY341495 on EPSC frequency (number/s) and amplitude (pA). All values displayed are means S.E.

LY341495 n Basal 5-HT LY341495 LY341495/5-HT

EPSC frequency30 nM (n 4) 4 5.1 0.9 34.7 13.7a 5.3 1.2 42.8 16.8100 nM (n 5) 5 5.4 1.3 18.6 6.2a 5.0 1.2 24.2 7.1b

1 M (n 6) 6 6.3 1.3 31.6 9.5a 4.8 1.4 45.3 10.4b

10 M (n 5) 5 5.4 1.4 18.8 5.1a 7.6 3.6 31.3 9.3b

30 M (n 4) 4.9 1.3 21.1 7.8a 4.4 1.9 34.6 10.0b

EPSC amplitude30 nM 4 67.4 5.0 83.1 7.8 63.4 6.5 86.9 8.8100 nM 5 78.7 3.9 84.8 4.4 79.7 6.7 95.3 6.7b

1 M 6 57.8 3.9 81.1 7.7a 58.2 5.2 91.9 10.0b

10 M 5 70.6 8.5 81.4 5.6 77.1 6.0 98.3 5.4b

30 M 4 68.2 3.5 80.0 4.9a 68.1 8.6 92.1 6.9b

a P .05 compared to basal value.b P .05 compared to 5-HT value. Note that there were no significant differences between the basal value compared to the respective LY341495 value.



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Changes in the frequency of synaptic currents is generallyattributed to a presynaptic locus, whereas changes in theamplitude of synaptic currents can be attributed to either apresynaptic or postsynaptic locus (Van der Kloot, 1991). Fur-thermore, LY354740 did not suppress the inward currentinduced by bath application of AMPA following blockade ofsynaptic transmission by a combination of TTX and Ca2-free ACSF. Effects of the mGlu2/3 agonists at presynapticsites would be consistent with evidence suggesting that acti-

vation of either mGlu2 or mGlu3 receptors suppresses gluta-mate release at corticostriatal synapses (Lovinger and Mc-Cool, 1995).

At this time, however, we cannot rule out the possibilitythat the mGlu agonists and antagonists might have postsyn-aptic effects to account for the alteration in 5-HT-inducedEPSC frequency and amplitude. The apical dendrites containNa and Ca2 conductances that are thought to play a dra-matic role in the amplification of distal synaptic signals

Fig. 3. LY354740: suppression ofsynaptic currents/potentials. A,the effects of 5-HT; then the ef-fects of the mGlu2/3 agonistLY354740 on 5-HT-induced EP-

SCs in the same cell. The com-mand potential in this cell was setat 70 mV. B, the effect of LY354740 on electrically evokedEPSPs after DOI treatment (3M). This cell was held at 80mV during the entire experiment.C, summary for LY354740 on thenormalized frequency of 5-HT-in-duced spontaneous EPSCs, theamplitude of electrically evokedearly EPSPs, and the amplitudeof the electrically evoked late EP-SPs after DOI treatment.



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(Schiller et al., 1997; Schwindt and Crill, 1997). Activation ofgroup II mGlu receptors in the apical dendritic compartmentof pyramidal cells, despite the small and inconsistent out-ward currents observed in somatic recordings, might sup-press these Na and Ca2 conductances either directly viaeffects on Ca2 conductances or indirectly via effects on K

conductances. Thus, the effect of mGlu agonists could be onthe apical dendrite of pyramidal cells that were being re-corded. Alternatively, the effect of mGlu agonist could be onthe apical dendrite of neighboring pyramidal cells of theneuron from which the recording was taken. However, nei-

ther scenario appears to account for the selective suppressionof 5-HT-induced EPSCs by -opioid agonists, which suggestsa subcortical source for the afferents on which 5-HT inducesexcitatory amino acid release (Marek and Aghajanian,1998a). An increase in impulse flow within intracortical cir-cuitry between neighboring pyramidal cells also fails to ac-count for the 60% block of 5-HT-induced EPSCs by chemi-cal lesions of the medial thalamus (Marek and Gewirtz,1999). Previously, we have found that the hot spots forinduction of EPSCs by microiontophoretic application of5-HT are restricted to the apical dendrites of neocorticallayer V pyramidal cells in layers I and Va. This suggests that5-HT induces EPSCs through a focal action that does notrequire impulse flow (see Aghajanian and Marek, 1999b).These hot spots correspond to the laminae that are rich in5-HT-containing axons and 5-HT2A receptors (Blue et al.,1988; Aghajanian and Marek, 1997). By inference, these con-siderations suggest that mGlu2/3 receptors play a particu-larly important role in the integration of synaptic activity byapical dendrites of the layer V pyramidal cells.

As an initial step to determine the underlying neuroana-tomical basis for the interactions between mGlu2/3 and

5-HT2A receptors, we used autoradiography of coronal sec-tions including the medial prefrontal cortex with[3H]LY354740 and [125I]DOI as ligands for these receptors,respectively. We observed a striking similarity in the laminardistribution of mGlu2/3 binding and 5-HT2A binding in themedial prefrontal cortex. Previously, the highest density of5-HT2A receptors in the neocortex and transitional cortex hasbeen reported to be in layers I and Va (Blue et al., 1988).Similarly, the heaviest density of mGlu2/3 receptor bindingin the medial prefrontal cortex also was in layers 1 and Va. Incontrast, the peak mGlu2/3 binding in the Fr1 and Par1regions of the frontoparietal cortex was present in layersIIIV, superficial to the heaviest density of DOI binding inlayer Va. In this context, the lower potency of LY354740 in

suppressing 5-HT-induced EPSCs in the frontoparietal re-gion compared with the medial prefrontal cortex might beattributed to a lower density of mGlu2/3 receptors on thenerve endings innervating the apical dendrites of layer Vpyramidal cells in the former region. Additional work will benecessary to determine whether mGlu2/3 and 5-HT2A recep-tors are localized in the same nerve terminals in the medialprefrontal cortex and other cortical regions.

The pattern of [3H]LY354740 binding in the medial pre-frontal cortex appears similar to immunostaining for mGlu2receptors from coronal sections including the medial prefron-tal cortex (Ohishi et al., 1997). This may provide an initialsuggestion as to the role of mGlu2 versus mGlu3 receptors inmediating the effects reported in this paper. LY354740, butnot LY379268, was 3- to 5-fold more selective at suppressing5-HT-induced EPSCs compared with electrically evokedearly EPSPs. This corresponds to an 5-fold selectivity forLY354740 at mGlu2 versus mGlu3 receptors in suppressingactivation of adenylyl cyclase (Table 1). Interestingly, boththe protein and mRNA for mGlu2 is found throughout thethalamus, especially in the midline and intralaminar nucleithat are known to project to prefrontal cortex (Berendse andGroenewegen, 1991; Ohishi et al., 1993a, 1997). In prelimi-nary studies, we have found that chemical medial thalamiclesions result in a significant reduction of 5-HT-induced EP-SCs in the medial prefrontal cortex (Marek and Gewirtz,

Fig. 4. A mGlu2/3 antagonist blocks the suppressant activity ofLY354740. The mGlu2/3 antagonist LY341495 (1 M) results in a right-ward shift in the concentration response curve for the suppression of5-HT-induced EPSCs by LY354740, resulting in a pA

2value of 7.27 (top).

The EC50 for LY354740 was 89 nM (n 5); in a separate group of cells inthe presence of LY341495, the EC

50for LY354740 was 1733 nM (n 5).

LY341495 (1 M) also results in a rightward shift in the concentrationresponse curve for the suppression of electrically evoked early EPSPs byLY354740 (bottom). The electrical stimulus was adjusted in these exper-iments to evoke an early EPSP at 70% of the amplitude necessary toelicit a spike (e.g., 13.2 2.7 and 13.8 1.6 mV, respectively). The EC

50

for LY354740 was 454 nM (n 4); in a separate group of cells in thepresence of LY341495, the EC

50for LY35470 was 36,664 nM (n 4) to

yield a pA2 value of 7.9. LY354740 was tested in separate groups of cellsin the absence and presence of LY341495 because the slow washout ofLY354740 from the slices made a within cell design unpractical.



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1999). In contrast to mGlu2, the presence of mGlu3 receptormRNA in the thalamus appears largely restricted toGABAergic cells in the reticular nucleus of the thalamus(Ohishi et al., 1993b; Petralia et al., 1996) that do not projectto the neocortex. This differential localization suggests thatthe suppression of 5-HT-induced spontaneous EPSCs by thegroup II mGlu agonists may be mediated mainly via activa-tion of mGlu2. On the other hand, mRNA for mGlu3 appearsto be present in almost all pyramidal cells of the neocortex.Thus, activation of mGlu3 by the group II mGlu receptoragonists could mediate a significant component of the sup-pression of early evoked EPSPs (in layer V pyramidal cells) toelectrical stimulation of the forceps minor that would exciteprimarily corticofugal fibers arising from both layer V and

VI. At this time, the factor that accounts for the 3- to 5-fold

selectivity of LY354740 for suppression of 5-HT-induced EPSCsversus electrically evoked early EPSPs (Figs. 3 and 4; Table 3)remains to be explained. Several recent studies have found 4-to 5-fold selectivity for LY354740 in suppressing the inhibitionof adenylyl cyclase in cell lines expressing mGlu2 versus mGlu3receptors (Schoepp et al., 1997; Monn et al., 1999). In contrast,the mGlu2/3 antagonist LY341495, which is 3-fold less potent

at mGlu2 versus mGlu3 receptors, was 4-fold less potent atsuppressing 5-HT-induced EPSCs than electrically evokedearly EPSPs. These considerations suggest that group II mGluagonists might suppress 5-HT-induced EPSCs and electricallyevoked early EPSPs via activation of mGlu2 and mGluR3, re-spectively. However, LY379268 lacked selectivity in suppress-ing electrically evoked early EPSPs versus 5-HT-induced EP-SCs. A recent report found that this mGlu agonist was slightlyless than 2-fold selective in suppressing adenylyl cyclase activ-ity in cell lines expressing mGlu2 versus mGlu3 receptors(Monn et al., 1999). The relationship between the functionalactivity of these mGlu receptors in cell lines versus in nativetissue remains to be determined. It is clear that definitiveidentification of the receptors involved in these responses re-quires agonists/antagonists with greater selectivity or the use oftransgenic mice with disruption of mGlu2 or mGlu3 receptors.

Clinical applications for the mGlu2/3 agonist LY354740have previously been raised for the treatment of anxietydisorders, nicotine withdrawal, and schizophrenia (Heltonet al., 1997, 1998; Moghaddam and Adams, 1998). Addi-tional clinical implications of a physiological interactionbetween 5-HT2A and mGlu receptors in the medial prefron-tal cortex is intriguing because the medial prefrontal cor-

TABLE 3

Affinity of the mGlu2/3 agonists LY354740, LY379268 and the mGlu2/3 antagonist LY341495 at both cloned human (h) metabotropic glutamatereceptors (binding studies and functional assays) and rat forebrain homogenates compared with functional effects on 5-HT-induced EPSCs andearly and late components of the electrically evoked EPSPs following DOI treatment.

Mean S.E. (nM)

LY354740 LY379268 LY341495

Inhibition of forskolin-activated adenylyl cyclaseHuman mGlu2 (EC50) 5.1 0.3

a 2.7 0.3a

Human mGlu3 (EC50) 24.3 0.5a 4.6 0.1a

5-HT-induced EPSCs and evoked EPSPs5-HT-induced EPSCsb 89 30 (EC50) 231 54 (EC50) 54 7.0 (Kb)Evoked late EPSPs after DOIc 85 22 (EC

50) 260 151 (EC

50) N.D.

Evoked early EPSPsd 235 108 (EC50) 276 84 (EC50) 13 6.7 (Kb)

N.D., not determined.a Potency (EC50, nM) for suppression of forskolin-activated adenylyl cyclase for LY35740 (Schoepp et al., 1997) and LY379268 (Monn et al., 1999).b,c,d EC50 values (nM) for agonists at suppressing 5-HT-induced EPSCs, evoked late EPSCs after DOI, and evoked early EPSPs. Apparent Kb value of antagonist for

blocking suppressant action of LY354740 (nM) as determined from pA 2 value.

Fig. 5. A, LY354740 does not alter thesteady state inward current induced by

AMPA following block of synaptictransmission. An example of the rela-tive lack of suppression by LY354740 (1M, 5 min pretreatment) of the steady-state inward current induced by AMPA(5 M, 45 s) in a layer V medial prefron-tal pyramidal cell following bath perfu-sion of TTX (2 M) to block impulseflow. The command potential was set at70 mV in this cell. B, an EPSC ampli-tude cumulative frequency plot in a sin-gle cell for the effect of 5-HT versus acombination of 5-HT and a concentra-tion of LY354740 that decreased theEPSC frequency by 50%.



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tex is believed to be involved in the pathophysiology ofmood disorders and schizophrenia (Kroeze and Roth, 1998;Marek and Aghajanian, 1998b). Indeed, most of the atyp-ical antidepressants (e.g., mirtazepine, mianserin, nefaz-odone, trazodone, and iprindole) share 5-HT2A antagonism

as their most potent common pharmacological action(Richelson and Nelson, 1984; Wander et al., 1986; Eison etal., 1990; Marek et al., 1992; de Boer, 1996). Clozapine andthe newest generation of atypical antipsychotics (e.g.,olanzepine, risperidone, and the putative antipsychotic

Fig. 6.Autoradiography of [3H]LY354740

binding (top) and [125I]DOI binding (bot-tom) after subtraction of backgroundbinding in a coronal slice through medialprefrontal cortex (transitional cortex) andfrontoparietal neocortex at a level corre-sponding to the physiological studies.Note the superficial and midcortical in-crease in [125I]DOI binding, which isknown to be present in layers I and Va ofthe neocortex and transitional cortex. Thelaminar distribution of [3H]LY354740binding and [125I]DOI binding was simi-lar in the medial prefrontal cortex, butnot in the frontoparietal cortex.



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M100,907) also share potent 5-HT2A antagonism (Aghaja-nian and Marek, 1999b) that may contribute to the allevi-ation of positive and/or negative symptoms of schizophre-nia. Future clinical investigations with the mGlu agonistsin the treatment of depression and schizophrenia will be ofinterest.

Acknowledgments

We thank Nancy Margiotta for technical assistance and LeslieRosello for secretarial assistance.

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Fig. 7. Histogram of [3H]LY354740 binding (top) and[125I]DOI binding(bottom) in medial prefrontal cortex and frontoparietal cortex. The dataare presented in fmol of ligand bound/mg protein (mean S.E.). Sixsections were obtained from each of four rats to calculate the averageddata. The increased density of 5-HT

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Send reprint requests to: Dr. Gerard J. Marek, Yale School of Medicine,Department of Psychiatry, Connecticut Mental Health Center and the RibicoffResearch Facility, 34 Park St., New Haven, CT 06508. E-mail: [email protected]


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