0022-3565/00/2921-0076$03.

00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics JPET 292:76 87, 2000

Vol. 292, No. 1 Printed in U.S.A.

Physiological Antagonism between 5-Hydroxytryptamine2A and Group II Metabotropic Glutamate Receptors in Prefrontal Cortex1
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 Mental Health 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) receptors have been linked to the action of hallucinogens and atypical antidepressant/antipsychotic drugs. Previously, we have shown in cortical layer V pyramidal cells that a nonselective metabotropic glutamate (mGlu) receptor agonist suppresses the induction of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) via activation of 5-HT2A receptors. In this study, we tested the ability of the selective mGlu2/3 agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and the selective mGlu2/3 antagonist 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) to modulate serotonin(5-HT)-induced EPSPs and electrically evoked EPSPs by using intracellular recording from layer V pyramidal cells in medial prefrontal cortex. The mGlu2/3 antagonist LY341495 increased the frequency and amplitude of 5-HT-induced EPSCs, suggesting a role for mGlu2/3 receptors in mediating the action of endoge-

nous glutamate on autoreceptors. Conversely, the mGlu2/3 agonist LY354740 was highly effective and potent (EC50 89 nM) in suppressing glutamate release induced by 5-HT2A receptor activation in the medial prefrontal cortex, probably via a presynaptic mechanism. The mGlu2/3 antagonist LY341495 potently blocked the suppressant effect of LY354740 on 5-HTinduced EPSCs as well as electrically evoked early EPSPs. Autoradiography with the radioligands [3H]LY354740 and [125I]( )-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane shows a striking overlap of the laminar distribution of mGlu2/3 and 5-HT2A receptors in the medial prefrontal cortex that is not apparent in other cortical regions. These findings suggest a close coupling between mGlu2/3 and 5-HT2A receptors in the prefrontal cortex that may be relevant for novel therapeutic approaches in the treatment of neuropsychiatric syndromes such as depression and schizophrenia.

5-Hydroxytryptamine2A (5-HT2A) receptor antagonists block the psychotomimetic effects of hallucinogens in humans (Vollenweider et al., 1998) and are thought to contribute to the therapeutic effects of atypical antidepressant/antipsychotic drugs (Kroeze and Roth, 1998; Marek and Aghajanian, 1998b). Furthermore, increased glutamate release in the prefrontal cortex appears to be a common feature

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 (National Institute of Mental Health; G.K.A.), a National Alliance for Research on Schizophrenia and Depression (NARSAD) 1999 Fairfax Investigator Award (G.J.M.), the State of Connecticut (G.J.M., G.K.A.), and Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN.

shared by both noncompetitive N-methyl-D-aspartate antagonists and hallucinogenic drugs (Aghajanian and Marek, 1999b), both of which mimic some of the symptoms of acute psychosis. 5-HT2A receptor activation increases the frequency of spontaneous (nonelectrically evoked) excitatory postsynaptic currents (EPSCs) in apical dendrites of neocortical layer V pyramidal cells in a novel manner, suggesting focal release of glutamate from discrete pathways (Aghajanian and Marek, 1997). This induction of an increase in the frequency of EPSCs by serotonin (5-HT) is completely blocked by the -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist LY293558 (Aghajanian and Marek, 1997). The ability of 5-HT2 antagonists to block 5-HT-induced EPSCs is clearly mediated through 5-HT2A,

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-1yl)-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-dimethoxy-4-iodophenyl)-2-aminopropane; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionate; TTX, tetrodotoxin; ACSF, artificial cerebrospinal fluid. 76

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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-dimethoxy4-iodophenyl)-2-aminopropane (DOI), a hallucinogenic drug, induces an increase in the frequency of EPSCs, reaching only 10% of the level for 5-HT itself. However, DOI induces an increase in the late component of electrically evoked EPSCs, an effect which is blocked by the selective 5-HT2A antagonist and putative antipsychotic drug M100,907 (Aghajanian and Marek, 1999a). We have previously suggested that a common mechanism of glutamate release may underlie both the increased frequency of 5-HT-induced EPSCs and electrically evoked late EPSPs occurring after DOI application (Aghajanian and Marek, 1999a). This common mechanism may involve the asynchronous pathway of glutamate release because addition of Sr2 to a Ca2 -free artificial cerebrospinal fluid (ACSF) supported 5-HT-induced EPSCs under conditions in which the synchronous pathway of glutamate release was blocked (Goda and Stevens, 1994; Aghajanian and Marek, 1999a). Furthermore, addition of Sr2 to a Ca2 -free ACSF also supported electrically evoked late EPSCs under conditions in which the evoked early EPSCs, via the synchronous pathway of glutamate release (Goda and Stevens, 1994), were blocked. The electrically evoked late EPSCs after DOI application appeared similar to the electrically evoked late EPSCs from the Sr2 substitution experiments. Whether the electrically evoked late EPSCs represent asynchronous release of transmitter or conventional polysynaptic EPSCs, agents that suppress prefrontal glutamate release induced by activation of 5-HT2A receptors could provide a novel therapeutic approach to the treatment of depression and schizophrenia (Marek and Aghajanian, 1998b). One possible approach to suppressing glutamate release is through metabotropic glutamate (mGlu) receptors because many subtypes function as presynaptic autoreceptors on glutamatergic terminals (Conn and Pin, 1997). The mGlu receptors are a novel family of glutamate G-protein coupled receptors that are commonly separated into three classes based on both pharmacology and signal transduction pathways (Conn and Pin, 1997; Schoepp et al., 1999). Group I mGlu receptors (e.g., mGlu1 and mGlu5) are coupled to phospholipase C and phosphoinositide 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 formation and are thought to function as inhibitory presynaptic autoreceptors that may play a role in synaptic plasticity (Conn and Pin, 1997; Li et al., 1998). These group II and group III mGlu receptors have overlapping, but distinct, patterns 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 agonist, (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 200 M), was found to suppress 5-HT-induced EPSCs (Aghajanian and Marek, 1997). Recently, novel conformationally constrained analogs of glutamate that are selective for the group II mGlu receptors at low concentrations, including two agonists, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6dicarboxylate monohydrate (LY354740) and ( )-2-oxa-4aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), and an antagonist, (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)3(xanthy-9-yl)propanoic acid (LY341495), have been developed (Monn et al., 1997, 1999; Schoepp et al., 1997; Fitzjohn

et al., 1998; Kingston et al., 1998). We now provide evidence that physiological as well as pharmacological activation of mGlu2/3 receptors suppresses glutamate release induced by 5-HT2A receptor activation in the medial prefrontal cortex (transitional neocortex including both the prelimbic area of the medial prefrontal cortex and the anterior cingulate). Furthermore, a striking laminar overlap was found in the medial prefrontal cortex compared with other cortical regions for 5-HT2A and mGlu2/3 receptor binding that may be relevant to targeting drugs for neuropsychiatric syndromes involving the prefrontal cortex.

Materials and Methods

Autoradiography. Whole brains were obtained from adult male Sprague-Dawley rats (120 200 g), rapidly frozen in powdered dry ice, and mounted on cryostat chucks. Coronal sections (20 m) were cut from the prefrontal cortex and thaw-mounted on gelatin-coated slides. Sections were stored at 20C before use. For [3H]LY354740 binding, tissue sections were preincubated in ice-cold 10 mM potassium phosphate buffer with 100 mM potassium bromide (phosphate/ bromide buffer), pH 7.6, for 30 min to remove endogenous receptor ligands, then rapidly dried under a stream of cool air. Then the sections were incubated for 60 min at room temperature in phosphate/bromide buffer with 50 nM [3H]LY354740 (custom prepared by Amersham, Buckinghamshire, England). Nonspecific binding was determined using adjacent sections with 1 mM L-glutamate in the buffer solution. After incubation, each slide was rinsed rapidly three times with 2 ml of ice-cold phosphate/bromide buffer followed by a final rinse with 2 ml of ice-cold glutaraldehyde (49%)/acetone mixture (1:19, v/v), then quickly dried under a stream of warm air. Rinse time was 10 s per slide. Sections were opposed, with tritium microscales, to tritium-sensitive film (Hyperfilm-3H, Amersham, Piscataway, NJ) for 14 days. For measurement of 5HT2A binding sites, tissue sections from the same rats were incubated for 90 min at room temperature in a pH 7.4 buffer (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 by incubation of adjacent sections in buffer and radioligand with 1 M M100,907 (formerly known as MDL100,907) added. After incubation, the slides were rinsed twice for 10 min in ice-cold assay buffer followed 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-sensitive film (Amersham, Piscataway, NJ) for 30 h. Quantitative autoradiogram analysis was performed with a computer-assisted image analyzer (MCID; Imaging Research Inc., St. Catherines, Ontario). Optical density values were converted to femtomole per milligram of protein by using a computer generated regression analysis. Values displayed in Fig. 4 represent data averaged from four rats with six sections per rat. This data was analyzed with 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 et al., 1994). Cell membranes from human mGlu2- and human mGlu3expressing RGT cells (cells transfected with a rat glutamate transporter) were obtained as described previously (Johnson et al., 1999). Frozen tissue pellets were thawed on the day of assay and washed three times with ice-cold assay buffer (phosphate/bromide buffer). To start the reaction, rat tissue (0.15 0.20 mg of protein) or mGlu2 or mGlu3 tissue (0.04 0.06 mg of protein) was added to deep-well polypropylene microtiter plates or 5-ml scintillation vials (for rat brain tissue) that contained [3H]LY354740 (10 nM) and appropriate concentrations of test compounds in assay buffer. Final assay volume

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Vol. 292 for the mGlu antagonist LY341495 to effect a shift in the concentration-response curve for LY354740 (Arunlakshana and Schild, 1959). Compounds. The drugs used in this study were obtained from the following suppliers: Sigma Chemical Co. (St. Louis, MO; 5-HT creatine 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 monohydrate (LY366563), LY379268, and LY341495 were prepared by J.A.M. at the Lilly Research Laboratories (Indianapolis, IN).

was 0.5 ml. Nonspecific binding was defined with 1 mM L-glutamate. Samples were incubated on ice for 60 min and bound radioligand was separated from free radioligand by rapid filtration with washes with 1 ml of ice-cold assay buffer using a Whatman GF-B filter for mGlu2 or mGlu3 binding. For rat brain tissue, bound [3H]LY354740 was separated from free [3H]LY354740 by centrifugation as described previously (Wright et al., 1994). Protein concentration was determined by using the Pierce Coomassie micro assay (Rockford, IL). Ki values were determined by using nonlinear regression in the GraphPad Prism program (GraphPad Software, Inc.). Electrophysiology. Brain slices were prepared from male Sprague-Dawley rats (120 200 g) as described previously (Aghajanian and Rasmussen, 1989). Briefly, rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and decapitated. Coronal slices (500 m) were cut with an oscillating-blade tissue slicer at a level corresponding to approximately 2.5 mm anterior to bregma (Paxinos and Watson, 1986). A slice containing the medial prefrontal cortex was then transferred to the stage of a fluid-gas interface chamber, which had a constant flow of humidified 95% O2, 5% CO2. The slices were perfused in a chamber heated to 34C with normal ACSF, which consisted of 126 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 26 mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM D-glucose. Intracellular recording and single-electrode voltage clamping were conducted in layer V pyramidal cells by using an Axoclamp-2A (Axon Instruments, Inc., Foster City, CA) as previously described (Aghajanian and Marek, 1997). Stubby electrodes ( 8 mm, shank to tip) with relatively low capacitance and resistance (30 60 M ) were filled with 1 M potassium acetate. The cells were voltage clamped at 70 mV. The EPSCs recorded under these conditions do not appear to be contaminated by reversed inhibitory postsynaptic currents for the following reasons. Only a small fraction of 5-HT-induced EPSCs ( 15%) are blocked by bicuculline during intracellular recordings using KCl-containing electrodes, suggesting the presence of some reverse inhibitory postsynaptic currents. In contrast, the 5-HT-induced EPSCs recorded with nonchloride-containing electrodes (i.e., potassium acetate or gluconate) at holding potentials near ECl are completely blocked by the AMPA/kainate antagonist LY293558 (Aghajanian and Marek, 1997). The voltage-clamp signals were lowpass filtered (1000 Hz) and data were acquired with a pCLAMP/ Digidata 1200 system (Axon Instruments, Inc.). EPSC frequencies were obtained from 10 successive episodes (1-s duration) during the baseline and drug treatment periods. Evoked potentials were obtained while holding cells at 80 mV and stimulating the forceps minor in the white matter deep in the cortex. EPSC frequency and amplitude were determined with Mini Analysis Program (www.synaptosoft.com; Synaptosoft, Inc., Leonia, NJ) using thresholds of 10 pA and an area of 150 fCs 1 for synaptic currents. Statistical comparisons of within-cell responses were made with two-tailed paired t-tests requiring P .05 for statistical significance. The Kolmogorov-Smirnov test was used to determine whether significant changes occurred in the EPSC amplitude cumulative probability plots. The determination of EC50 values for the suppression of 5-HTinduced increases in EPSC frequency or of evoked EPSPs were calculated by nonlinear curve fitting (DeltaGraph 4.0; DeltaPoint, Monterey, CA). The Schild equation was used to calculate pA2 values

Results
Binding Affinity of mGlu2/3 Agonists and Antagonist. The mGlu2/3 agonist LY354740 potently bound to human mGlu2 and mGlu3 receptors with a slight selectivity for mGlu2 compared with mGlu3 (Table 1; Fig. 1), consistent with a previous report of a 5-fold selectivity of LY354740 for mGlu2 versus mGlu3 in a functional assay (Schoepp et al., 1997). LY379268, another mGlu2/3 agonist, potently bound to human mGlu2 and mGlu3 receptors with a slight selectivity for mGlu2 versus mGlu3 (Table 1; Fig. 1), but selectivity for LY379268 at mGlu2 versus mGlu3 receptors has not been observed 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 nanomolar potency to both mGlu2 and mGlu3, but with 3-fold selectivity for mGlu3 versus mGlu2. All three mGlu2/3 ligands fully displaced [3H]LY354740 from rat forebrain binding sites with nanomolar potency (Fig. 1). Importantly, the rank order of potency for all three mGlu2/3 ligands at displacing the radioligand from the rat forebrain binding sites was similar to that found with human mGlu2/3 receptors transfected into the RGT cells. Electrophysiological Characteristics of Cortical Layer V Pyramidal Cells. Layer V pyramidal cells were recorded in a zone ca. 1/2 to 2/3 the distance between the pial surface and the subcortical white matter. The pyramidal cells in the present study had the following characteristics: resting potential, 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, except a single cell with an intrinsically bursting firing pattern to a constant depolarizing pulse, had the previously reported characteristics (McCormick et al., 1985; Connors and Gutnick, 1990) of regularly spiking pyramidal cells. The mGlu2/3 Antagonist LY341495 Enhances the Frequency and Amplitude of 5-HT-Induced EPSCs. We had previously observed that the AMPA/kainate antagonist LY293558 and the nonselective mGlu agonist (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) completely blocked 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 brain homogenates of [3H]LY354740 binding. All values displayed are means S.E. (n 3).
hmGlu2 Compound KI nM 95% CI KI nM 95% CI KI nM 95% CI hmGlu3 Rat forebrain

LY354740 LY379268 LY341495

47.8 6.65 6.01

15 1.3 1.3

24.764.4 4.039.08 3.409.96

65.8 12.8 2.52

27 5.3 0.6

66.2146 5.7316.9 1.393.97

21.4 1.45 0.68

2.0 0.18 0.1

17.227.5 0.852.08 0.311.08

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Fig. 1. Displacement of [3H]LY354740 binding by mGlu2/3 ligands. Each panel shows the displacement of [3H]LY35740 binding by the mGlu2/3 agonists LY354740 and LY379268, and the mGlu2/3 antagonist LY341495 in cells transfected with either human mGlu2 receptors (top), human mGlu3 receptors (middle), or to rat forebrain binding sites (bottom).

these drugs may be acting at a postsynaptic and a presynaptic site, respectively (Aghajanian and Marek, 1997). To test whether tonic release of endogenous glutamate might be acting on inhibitory autoreceptors to reduce the sustained increase 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 intracellular recordings from layer V pyramidal cells of the medial prefrontal cortex. It should be noted that LY341495 ( 1 M) does not block the suppressant action of the group III mGlu receptor agonist L-serine-O-phosphate (L-SOP) on 5-HT-induced EPSPs (unpublished observations, G.J.M.). The mGlu2/3 antagonist LY341495 (20 min) enhanced the frequency of 5-HT-induced EPSCs by 30 to 65% (0.130 M; Fig. 2; Table 2); a significant increase in the frequency of EPSCs was observed first at 100 nM, although a trend was present for a significant increase at 30 nM (P .09, n 4). The classical interpretation of a change in the frequency of synaptic currents is via an effect on the presynaptic terminal (Van der Kloot, 1991). LY341495 also increased the amplitude 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 with two or more concentrations), LY341495 significantly increased the EPSC amplitude for the 5-HT-induced EPSCs (Kolmogorov-Smirnov test, P .05 to P .0001, Fig. 2B). A change in the amplitude of synaptic currents could be mediated by either a presynaptic or postsynaptic effect (Van der Kloot, 1991). LY341495 did not alter the amplitude of electrically evoked early EPSPs (data not shown, n 2) or the amplitude of spontaneously occurring EPSCs (contrast Fig. 2, A1 and A3; Table 2) observed under basal conditions in 17 of the 19 cells tested (Kolmogorov-Smirnov test, P .05). Together, these results suggest that mGlu2 and/or mGlu3 function as autoreceptors that are tonically activated by physiological concentrations of glutamate released via 5-HT2A receptor activation. mGlu 2/3 Agonists Suppress 5-HT-Induced EPSCs and Electrically Evoked EPSPs. Intracellular recordings from layer V pyramidal cells of the medial prefrontal cortex were used to test mGlu2/3 agonists for both the suppression of 5-HT-induced EPSCs and the increase in electrically evoked late EPSPs after application of the 5-HT2A agonist DOI. LY354740 (Schoepp et al., 1997) was virtually equipotent at suppressing 5-HT-induced EPSCs (n 5) and DOIenhanced electrically evoked late EPSPs (n 5) (EC50 values: 89.1 versus 85.3 nM). In contrast, LY354740 was 3- to 5-fold less potent at suppressing electrically evoked early EPSPs (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 not suppress 5-HT-induced EPSCs in any of the four cells from the medial prefrontal cortex tested (data not shown). The suppressant effect of LY354740 on 5-HT-induced EPSCs showed regional variability in that neurons from the medial prefrontal cortex were 4-fold more sensitive to this drug than were a random sample of cells (n 3) from the Fr1, Fr3, and Par1 areas of the frontoparietal neocortex (Paxinos and Watson, 1986) (EC50 398 142, n 3). Another mGlu2/3 agonist, LY379268 (Table 3), was approximately equipotent in suppressing 5-HT-induced EPSCs and the electrically evoked early EPSPs and late EPSPs after DOI application (EC50 values: 230 276 nM, n 4). A Selective mGlu2/3 Antagonist Blocks the Effects of LY354740. A concentration of mGlu2/3 antagonist LY341495 that does not appear to block the suppressant action by the mGlu4/6/7/8 receptor agonist L-SOP on 5-HT-induced EPSPs ( 1 M, G.J.M., unpublished observations) was tested

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Fig. 2. A mGlu2/3 antagonist enhances 5-HT-induced synaptic currents. A, in this layer V pyramidal cell, the mean basal EPSC frequency and amplitude were 2.5 s 1 and 57.0 pA (A1). The mean 5-HT-induced EPSC frequency and amplitude were 31.3 s 1 and 72.7 pA (A2). The mean frequency and amplitude of EPSCs after a 20 min application of LY341495 (1 M) were 1.8 s 1 and 57.0 pA (A3). Note in A3 that LY341495 does not alter the frequency or amplitude of EPSCs from basal (A1) conditions. After LY341495 application in this cell, the 5-HT-induced EPSC frequency and amplitude was 61.1 s 1 and 99.6 pA (A4). All tracings were taken from the same cell, in which the command potential was set at 70 mV. B, an EPSC amplitude cumulative frequency plot in a single cell for the effect of 5-HT both before and after a 20 min treatment with LY341495. C, a summary of experiments (n 4 6) from 30 nM to 30 M LY341495. The mean 5-HT-induced EPSC frequencies and amplitudes (mean S.E.) for the basal, 5-HT, LY354740, and LY354740/5-HT conditions are displayed in Table 1. , significantly different from 5-HT alone, P .05. , significantly different from 5-HT alone, P .01.

against the suppressant action of the mGlu2/3 agonist LY354740. LY341495 resulted in a robust rightward shift in the concentration-response curve for the suppression by LY354740 against the frequency of 5-HT-induced EPSCs, yielding a pA2 value in the nanomolar range (pA2 7.27, n 5, Fig. 4). LY341495, which is 3-fold more potent at mGlu3 than mGlu2 receptors, was 4-fold more potent at blocking the suppressant effect of LY354740 on electrically evoked early EPSPs (pA2 7.9, n 5, Fig. 4) than on 5-HT-induced EPSCs (Table 1). The antagonism by LY341495 against suppression by LY354740 for both the 5-HT-induced EPSCs and the electrically evoked early EPSPs was surmountable by higher concentrations of LY354740, which is consistent with competitive antagonism. The potency of LY341495 in blocking the suppressant action of LY354740 on electrically evoked late EPSPs after DOI application was not tested.

Presynaptic versus Postsynaptic Action of LY354740. The 5-HT-induced EPSCs are mediated via AMPA/kainate receptors on layer V pyramidal cells because they are completely blocked by the AMPA/kainate antagonist LY293558 (Aghajanian and Marek, 1997). To evaluate whether a postsynaptic action could be involved in the suppression by mGlu agonists of the 5-HT-induced EPSCs, the effect of LY354740 on inward currents induced by bath application of AMPA (5 M) was examined in layer V pyramidal cells. After treatment of the slice with TTX (2 M) to block impulse flow, LY354740 did not block AMPA-induced inward currents (AMPA, 243 74 pA; LY354740 and AMPA, 233 75 pA; n 4; Fig. 5). LY354740 did induce small outward currents in two of the four cells after TTX treatment (40 and 60 pA). The effects of LY354740 and AMPA after TTX treatment were similar when the experiment was repeated by

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S.E.

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

EPSC frequency 30 nM (n 4) 100 nM (n 5) 1 M (n 6) 10 M (n 5) 30 M (n 4) EPSC amplitude 30 nM 100 nM 1 M 10 M 30 M

a b

4 5 6 5 4 5 6 5 4

5.1 5.4 6.3 5.4 4.9 67.4 78.7 57.8 70.6 68.2

0.9 1.3 1.3 1.4 1.3 5.0 3.9 3.9 8.5 3.5

34.7 18.6 31.6 18.8 21.1 83.1 84.8 81.1 81.4 80.0

13.7a 6.2a 9.5a 5.1a 7.8a 7.8 4.4 7.7a 5.6 4.9a

5.3 5.0 4.8 7.6 4.4 63.4 79.7 58.2 77.1 68.1

1.2 1.2 1.4 3.6 1.9 6.5 6.7 5.2 6.0 8.6

42.8 24.2 45.3 31.3 34.6 86.9 95.3 91.9 98.3 92.1

16.8 7.1b 10.4b 9.3b 10.0b 8.8 6.7b 10.0b 5.4b 6.9b

P P

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

applying the drugs in ACSF containing no Ca2 (n 2, data not shown). A more direct test of whether the suppressant effect of LY354740 on 5-HT-induced EPSCs was mediated via a presynaptic site involved analyzing the cumulative probability distributions of the EPSC amplitudes. In four of five cells, a concentration of LY354740 at approximately the EC50 for reducing the frequency of 5-HT-induced EPSCs did not cause a significant change (P .05) in the EPSC amplitude as assessed with the Kolmogorov-Smirnov test (Fig. 5B). The selective blockade in the frequency rather than the amplitude of 5-HT-induced EPSCs also argues for a presynaptic locus of action for LY354740 (Van der Kloot, 1991). [125I]DOI and [3H]LY354740 Autoradiography. To begin an examination of the neuroanatomical basis of the interaction between 5-HT2A receptors and the group II mGlu (mGlu2 and mGlu3) receptors, we compared the autoradiographic pattern of binding for [125I]DOI and [3H]LY354740 (Schoepp et al., 1997) in coronal brain sections including the medial prefrontal cortex by using concentrations of radioactive and displacement ligands to specifically label 5-HT2A and mGlu2/3 receptors, respectively (Figs. 6 and 7). Both [125I]DOI and [3H]LY354740 bound to the superficial and mid-layer of the medial prefrontal cortex. The laminar distribution of [125I]DOI is consistent with previous observations for peaks in specific 5-HT2A receptor binding in layers I and Va of the neocortex and transitional cortex (Blue et al., 1988). In all areas of the neocortex and transitional neocortex, the [3H]DOI binding was highest in layer Va and significantly lower in the intermediate layers (layers IIIV). In contrast to the medial prefrontal cortex where [3H]LY354740 binding was significantly lower in layers II/III than in layer Va, in the frontoparietal region (e.g., Par1), [3H]LY354740 binding was significantly higher in layers II/III/IV than in layer Va.

Discussion
The most striking finding from the present studies was that, in the absence of an exogenous agonist, the potent group II mGlu antagonist LY341495 increased the frequency and amplitude of 5-HT-induced EPSCs. This increase occurred at concentrations of LY341495 ( 1 M) that do not block the suppressant action on 5-HT-induced EPSCs by the group III mGlu agonist L-SOP. The mGlu antagonist did not alter the amplitude of either the basal EPSCs or the electri-

cally evoked early EPSPs, these being situations where no tonic activation of presynaptic autoreceptors by endogenous glutamate would be expected. However, when a vigorous and sustained increase in EPSCs occurred during the application of 5-HT, the frequency and amplitude of synaptic currents was almost always enhanced by the mGlu antagonist. These findings are consistent with the hypothesis that mGlu2/3 receptors function as inhibitory autoreceptors in cortical glutamatergic terminals whose transmitter release is positively regulated by 5-HT2A receptor activation. These observations appear to be similar to the previous demonstration in the hippocampus of a use-dependent activation of presynaptic mGlu receptors that function to decrease excitatory amino acid release (Scanziani et al., 1997). Conversely, mGlu2/3 agonists suppress glutamate release induced by 5-HT2A receptor activation from nerve endings that terminate onto cortical layer V pyramidal cells. Two potent and selective mGlu2/3 agonists, LY354740 (Monn et al., 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 induced by the partial 5-HT2A agonist DOI with a similar potency. This action was not shared by LY366553, the inactive ( )-isomer of LY354740. Furthermore, the selective mGlu2/3 antagonist LY341495 blocked the suppressant action of LY354740 on 5-HT-induced EPSCs or electrically evoked early EPSPs with potencies consistent with pharmacological antagonism of mGlu2/3 receptors (Kingston et al., 1998). Although both mGlu2/3 agonists LY354740 and LY379268 suppressed 5-HT-induced EPSCs, the 3-fold greater potency of LY354740 over LY379268 is surprising in light of the greater affinity of LY379268 over LY354740 at mGlu2, mGlu3, and rat forebrain binding sites. In this context, it should be noted that LY354740 and LY379268 are conformationally constrained analogues of glutamic acid (Monn et al., 1997, 1999). Perhaps differences in the potency of these agonists in receptor binding assays compared with functional assays in tissue slices could reflect differences in the ability of these compounds to access the receptor due to differing affinities at other synaptic sites such as glutamate transporters. The mGlu2/3 agonists appear to act on a presynaptic site to decrease glutamate release. In four of five cells, LY354740 did not alter the amplitude of 5-HT-induced spontaneous EPSCs at a concentration that decreased frequency by 50%.

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Fig. 3. LY354740: suppression of synaptic currents/potentials. A, the effects of 5-HT; then the effects of the mGlu2/3 agonist LY354740 on 5-HT-induced EPSCs in the same cell. The command potential in this cell was set at 70 mV. B, the effect of LY354740 on electrically evoked EPSPs after DOI treatment (3 M). This cell was held at 80 mV during the entire experiment. C, summary for LY354740 on the normalized frequency of 5-HT-induced spontaneous EPSCs, the amplitude of electrically evoked early EPSPs, and the amplitude of the electrically evoked late EPSPs after DOI treatment.

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

vation of either mGlu2 or mGlu3 receptors suppresses glutamate release at corticostriatal synapses (Lovinger and McCool, 1995). At this time, however, we cannot rule out the possibility that the mGlu agonists and antagonists might have postsynaptic effects to account for the alteration in 5-HT-induced EPSC frequency and amplitude. The apical dendrites contain Na and Ca2 conductances that are thought to play a dramatic role in the amplification of distal synaptic signals

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Fig. 4. A mGlu2/3 antagonist blocks the suppressant activity of LY354740. The mGlu2/3 antagonist LY341495 (1 M) results in a rightward shift in the concentration response curve for the suppression of 5-HT-induced EPSCs by LY354740, resulting in a pA2 value of 7.27 (top). The EC50 for LY354740 was 89 nM (n 5); in a separate group of cells in the presence of LY341495, the EC50 for LY354740 was 1733 nM (n 5). LY341495 (1 M) also results in a rightward shift in the concentration response curve for the suppression of electrically evoked early EPSPs by LY354740 (bottom). The electrical stimulus was adjusted in these experiments to evoke an early EPSP at 70% of the amplitude necessary to elicit a spike (e.g., 13.2 2.7 and 13.8 1.6 mV, respectively). The EC50 for LY354740 was 454 nM (n 4); in a separate group of cells in the presence of LY341495, the EC50 for LY35470 was 36,664 nM (n 4) to yield a pA2 value of 7.9. LY354740 was tested in separate groups of cells in the absence and presence of LY341495 because the slow washout of LY354740 from the slices made a within cell design unpractical.

(Schiller et al., 1997; Schwindt and Crill, 1997). Activation of group II mGlu receptors in the apical dendritic compartment of pyramidal cells, despite the small and inconsistent outward currents observed in somatic recordings, might suppress these Na and Ca2 conductances either directly via effects on Ca2 conductances or indirectly via effects on K conductances. Thus, the effect of mGlu agonists could be on the apical dendrite of pyramidal cells that were being recorded. Alternatively, the effect of mGlu agonist could be on the apical dendrite of neighboring pyramidal cells of the neuron from which the recording was taken. However, nei-

ther scenario appears to account for the selective suppression of 5-HT-induced EPSCs by -opioid agonists, which suggests a subcortical source for the afferents on which 5-HT induces excitatory amino acid release (Marek and Aghajanian, 1998a). An increase in impulse flow within intracortical circuitry between neighboring pyramidal cells also fails to account for the 60% block of 5-HT-induced EPSCs by chemical lesions of the medial thalamus (Marek and Gewirtz, 1999). Previously, we have found that the hot spots for induction of EPSCs by microiontophoretic application of 5-HT are restricted to the apical dendrites of neocortical layer V pyramidal cells in layers I and Va. This suggests that 5-HT induces EPSCs through a focal action that does not require impulse flow (see Aghajanian and Marek, 1999b). These hot spots correspond to the laminae that are rich in 5-HT-containing axons and 5-HT2A receptors (Blue et al., 1988; Aghajanian and Marek, 1997). By inference, these considerations suggest that mGlu2/3 receptors play a particularly important role in the integration of synaptic activity by apical dendrites of the layer V pyramidal cells. As an initial step to determine the underlying neuroanatomical basis for the interactions between mGlu2/3 and 5-HT2A receptors, we used autoradiography of coronal sections including the medial prefrontal cortex with [3H]LY354740 and [125I]DOI as ligands for these receptors, respectively. We observed a striking similarity in the laminar distribution of mGlu2/3 binding and 5-HT2A binding in the medial prefrontal cortex. Previously, the highest density of 5-HT2A receptors in the neocortex and transitional cortex has been reported to be in layers I and Va (Blue et al., 1988). Similarly, the heaviest density of mGlu2/3 receptor binding in the medial prefrontal cortex also was in layers 1 and Va. In contrast, the peak mGlu2/3 binding in the Fr1 and Par1 regions of the frontoparietal cortex was present in layers IIIV, superficial to the heaviest density of DOI binding in layer Va. In this context, the lower potency of LY354740 in suppressing 5-HT-induced EPSCs in the frontoparietal region compared with the medial prefrontal cortex might be attributed to a lower density of mGlu2/3 receptors on the nerve endings innervating the apical dendrites of layer V pyramidal cells in the former region. Additional work will be necessary to determine whether mGlu2/3 and 5-HT2A receptors are localized in the same nerve terminals in the medial prefrontal cortex and other cortical regions. The pattern of [3H]LY354740 binding in the medial prefrontal cortex appears similar to immunostaining for mGlu2 receptors from coronal sections including the medial prefrontal cortex (Ohishi et al., 1997). This may provide an initial suggestion as to the role of mGlu2 versus mGlu3 receptors in mediating the effects reported in this paper. LY354740, but not LY379268, was 3- to 5-fold more selective at suppressing 5-HT-induced EPSCs compared with electrically evoked early EPSPs. This corresponds to an 5-fold selectivity for LY354740 at mGlu2 versus mGlu3 receptors in suppressing activation of adenylyl cyclase (Table 1). Interestingly, both the protein and mRNA for mGlu2 is found throughout the thalamus, especially in the midline and intralaminar nuclei that are known to project to prefrontal cortex (Berendse and Groenewegen, 1991; Ohishi et al., 1993a, 1997). In preliminary studies, we have found that chemical medial thalamic lesions result in a significant reduction of 5-HT-induced EPSCs in the medial prefrontal cortex (Marek and Gewirtz,

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

Inhibition of forskolin-activated adenylyl cyclase Human mGlu2 (EC50) Human mGlu3 (EC50) 5-HT-induced EPSCs and evoked EPSPs 5-HT-induced EPSCsb Evoked late EPSPs after DOIc Evoked early EPSPsd

5.1 24.3 89 85 235

0.3a 0.5a 30 (EC50) 22 (EC50) 108 (EC50)

2.7 4.6 231 260 276

0.3a 0.1a 54 (EC50) 151 (EC50) 84 (EC50) 54 7.0 (Kb) N.D. 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 pA2 value.

Fig. 5. A, LY354740 does not alter the steady state inward current induced by AMPA following block of synaptic transmission. An example of the relative lack of suppression by LY354740 (1 M, 5 min pretreatment) of the steadystate inward current induced by AMPA (5 M, 45 s) in a layer V medial prefrontal pyramidal cell following bath perfusion of TTX (2 M) to block impulse flow. The command potential was set at 70 mV in this cell. B, an EPSC amplitude cumulative frequency plot in a single cell for the effect of 5-HT versus a combination of 5-HT and a concentration of LY354740 that decreased the EPSC frequency by 50%.

1999). In contrast to mGlu2, the presence of mGlu3 receptor mRNA in the thalamus appears largely restricted to GABAergic cells in the reticular nucleus of the thalamus (Ohishi et al., 1993b; Petralia et al., 1996) that do not project to the neocortex. This differential localization suggests that the suppression of 5-HT-induced spontaneous EPSCs by the group II mGlu agonists may be mediated mainly via activation of mGlu2. On the other hand, mRNA for mGlu3 appears to be present in almost all pyramidal cells of the neocortex. Thus, activation of mGlu3 by the group II mGlu receptor agonists could mediate a significant component of the suppression of early evoked EPSPs (in layer V pyramidal cells) to electrical stimulation of the forceps minor that would excite primarily 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 EPSCs versus electrically evoked early EPSPs (Figs. 3 and 4; Table 3) remains to be explained. Several recent studies have found 4to 5-fold selectivity for LY354740 in suppressing the inhibition of adenylyl cyclase in cell lines expressing mGlu2 versus mGlu3 receptors (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 at suppressing 5-HT-induced EPSCs than electrically evoked early EPSPs. These considerations suggest that group II mGlu agonists might suppress 5-HT-induced EPSCs and electrically evoked early EPSPs via activation of mGlu2 and mGluR3, respectively. However, LY379268 lacked selectivity in suppressing electrically evoked early EPSPs versus 5-HT-induced EPSCs. A recent report found that this mGlu agonist was slightly less than 2-fold selective in suppressing adenylyl cyclase activity in cell lines expressing mGlu2 versus mGlu3 receptors (Monn et al., 1999). The relationship between the functional activity of these mGlu receptors in cell lines versus in native tissue remains to be determined. It is clear that definitive identification of the receptors involved in these responses requires agonists/antagonists with greater selectivity or the use of transgenic mice with disruption of mGlu2 or mGlu3 receptors. Clinical applications for the mGlu2/3 agonist LY354740 have previously been raised for the treatment of anxiety disorders, nicotine withdrawal, and schizophrenia (Helton et al., 1997, 1998; Moghaddam and Adams, 1998). Additional clinical implications of a physiological interaction between 5-HT2A and mGlu receptors in the medial prefrontal cortex is intriguing because the medial prefrontal cor-

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Fig. 6. Autoradiography of [3H]LY354740 binding (top) and [125I]DOI binding (bottom) after subtraction of background binding in a coronal slice through medial prefrontal cortex (transitional cortex) and frontoparietal neocortex at a level corresponding to the physiological studies. Note the superficial and midcortical increase in [125I]DOI binding, which is known to be present in layers I and Va of the neocortex and transitional cortex. The laminar distribution of [3H]LY354740 binding and [125I]DOI binding was similar in the medial prefrontal cortex, but not in the frontoparietal cortex.

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

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

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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 data are presented in fmol of ligand bound/mg protein (mean S.E.). Six sections were obtained from each of four rats to calculate the averaged data. The increased density of 5-HT2A receptor binding that was found in layers I and layer Va of the neocortex and anterior cingulate (Blue et al., 1988) was used as a template to identify the different cortical layers from the cortical regions of interest (Paxinos and Watson, 1986). Note that the increased density of 5-HT2A receptor binding that was found in layers I and Va of the anterior cingulate appears to be contiguous with the prelimbic and infralimbic cortical areas. , significantly different from density of layer Va binding of the respective area, P .05. , significantly different from density of layer Va binding of the respective area, P .01.

M100,907) also share potent 5-HT2A antagonism (Aghajanian and Marek, 1999b) that may contribute to the alleviation of positive and/or negative symptoms of schizophrenia. Future clinical investigations with the mGlu agonists in the treatment of depression and schizophrenia will be of interest.
Acknowledgments

We thank Nancy Margiotta for technical assistance and Leslie Rosello for secretarial assistance.
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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 Ribicoff Research Facility, 34 Park St., New Haven, CT 06508. E-mail: Gerard. Marek@yale.edu