AMPA receptor involvement in 5-hydroxytryptamine2A receptor-mediated pre-frontal cortical excitatory synaptic currents and DOI-induced head shakes

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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 62 – 71 www.elsevier.com/locate/pnpbp

AMPA receptor involvement in 5-hydroxytryptamine2A receptor-mediated pre-frontal cortical excitatory synaptic currents and DOI-induced head shakes Ce Zhang 1 , Gerard J. Marek ⁎ Department of Psychiatry, Yale University School of Medicine, Ribicoff Research Facilities of the Connecticut Mental Health Center, New Haven, Connecticut, USA Received 21 May 2007; received in revised form 3 July 2007; accepted 4 July 2007 Available online 20 July 2007

Abstract Glutamate plays an important role in the psychotomimetic effects of both channel blocking N-methyl D-aspartate (NMDA) receptor antagonists and hallucinogenic drugs which activate 5-hydroxytryptamine2A (5-HT2A) receptors. Previous work suggested that activation of non-NMDA ionotropic glutamate receptors mediates the effects of 5-HT-induced excitatory post-synaptic potentials/currents (EPSPs/EPSCs) when recording from layer V pyramidal cells in the rat medial pre-frontal cortex (mPFC). However, those effects are mediated by either α-amino-3-hydroxy-5methylisoxazole-4-propionate (AMPA) or kainate receptors of the iGluk5 subtype. To test whether activation of AMPA receptors is sufficient to mediate 5-HT-induced EPSCs, a 2,3-benzodiazepine that selectively blocks AMPA receptors was assessed. This selective AMPA receptor antagonist potently suppressed 5-HT-induced EPSCs. Since phenethylamine hallucinogens induce head shakes by activating 5-HT2A receptors in the mPFC and this action is modulated by glutamate, we also examined whether selective blockade of AMPA receptors would suppress DOIinduced head shakes. As predicted, we found that selective blockade of AMPA receptors suppressed DOI-induced head shakes. Given evidence that activation of AMPA receptors is an important downstream effect for both channel blocking NMDA receptor antagonists and phenethylamine hallucinogens, we also tested multiple doses of DOI with a sub-anesthetic dose of MK-801. Synergistic action between these two classes of psychotomimetic drugs was demonstrated by MK-801 enhancing DOI-induced head shakes and locomotor activity. These findings expand the dependence of both channel blocking NMDA receptor antagonists and phenethylamine hallucinogens on enhancing extracellular glutamate. © 2007 Published by Elsevier Inc. Keywords: 5-HT2A receptors; AMPA receptor antagonists; Glutamate; Hallucinogens; LY300164; MK-801

1. Introduction Abbreviations: 5-HT2A, 5-hydroxytryptamine2A; LY300164 or Talampanel, (R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4,5-h) (2,3) benzodiazepine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; APV, D(−)-2-amino-5-phosphonopentoic acid; GYKI 52466-HCl, 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine; CPP, 3-R-2-carboxypiperazin-4-propyl-1-phosphonic acid; DOI, 1-(2,5-dimethoxy-4-iodophenyl)-2aminopropane; MK-801, dizocilpine; EPSPs, excitatory post-synaptic currents; mPFC, medial pre-frontal cortex; IC50, inhibitory concentration50; iGluK5, receptor subtype, ionotropic glutamatekainate5; mGlu receptors, metabotropic glutamate; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; LY293558, (3S,4aR,6R,8aR)6[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid; TTX, tetrodotoxin. ⁎ Corresponding author. Eli Lilly and Company, Lilly Research Laboratories, Neuroscience Discovery Research, Lilly Corporate Center, Mail Drop 0510, Indianapolis, IN 46285 USA. Tel.: +1 317 651 4776; fax: +1 317 276 7600. E-mail address: [email protected] (G.J. Marek). 1 Present address: Department of Neurobiology, Shanxi Medical University, 56# Zinjian South Road, Taiyuan, Shanxi 03001, Peoples' Republic of China. 0278-5846/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.pnpbp.2007.07.009

Both the channel blocking N-methyl D-aspartate (NMDA) receptor antagonists and “hallucinogenic” drugs which activate 5-HT2A receptors constitute two of the three primary pharmacological drug models used to model aspects of schizophrenia (Abi-Saab et al., 1998; Gouzoulis-Mayfrank et al., 2005; Javitt and Zukin, 1991; Vollenweider et al., 1997b; Vollenweider et al., 1998; Woolley and Shaw, 1954). Both of these classes of psychotomimetic drugs mediate their effects, at least in part, through activation of non-NMDA ionotropic glutamate receptors (Aghajanian and Marek, 1999). The channel blocking NMDA receptor antagonists phencyclidine (PCP), dizocilpine (MK-801), and ketamine increase extracellular glutamate in the medial pre-frontal cortex (mPFC), increase locomotor activity, impair working memory, and induce impulsivity (Higgins et al.,

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2003; Moghaddam et al., 1997; Moghaddam and Adams, 1998). Local administration of the competitive NMDA receptor antagonist 3-R-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) into the mPFC also induces impulsive behavior, locomotor hyperactivity and increases in extracellular glutamate in the mPFC (Carli et al., 2004; Murphy et al., 2005). These effects are blocked by the selective 5-HT2A receptor antagonist M100907 (Carli et al., 2004; Ceglia et al., 2004), appearing analogous to 5-HT2A receptor blockade also attenuating a variety of other effects of non-competitive NMDA receptor antagonists ranging from increases in immediate early gene expression to immobility in the forced swimming test (Corbett et al., 1999; Habara et al., 2001; Higgins et al., 2003; MaurelRemy et al., 1995). The psychotomimetic effects of channel blocking NMDA receptor antagonists are mediated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate receptors and are suppressed by mGlu2/3 receptor agonists consistent with an autoreceptor function for mGlu2/3 receptors (Cartmell et al., 1999; Cartmell et al., 2000; Hauber and Andersen, 1993; Moghaddam et al., 1997; Moghaddam and Adams, 1998). Similarly, phenethylamine and indolealkylamine hallucinogens increase extracellular glutamate in the mPFC, increase the frequency of head shakes and locomotor activity, and enhance impulsivity (Carli and Samanin, 1992; Gingrich et al., 1999; Koskinen et al., 2000; Muschamp et al., 2004; Schreiber et al., 1995). The (1-(2,5,dimethoxy-4-iodophenyl))-2-aminopropane (DOI)-induced head shakes are also suppressed by mGlu2/3 receptor agonists and/or enhanced by mGlu2/3 receptor antagonists consistent with an autoreceptor function (Gewirtz and Marek, 2000; Klodzinska et al., 2002). Since the effect of 5-HT2A receptor activation in the mPFC on excitatory synaptic currents/ potentials is mediated by blockade of either AMPA and/or a kainate iGluK5 receptors, we sought to determine whether AMPA receptor activation is sufficient to mediate these effects and then to extend these findings to the behavioral effects of a phenethylamine hallucinogen. Therefore, we compared the effects of selective AMPA receptor antagonists from a 2,3-benzodiazepines structural class with the effects of a dihydroquinolinone AMPA/kainate receptor antagonist using 5-HT-induced EPSCs and DOI-induced head shakes as electrophysiological and behavioral measures of 5-HT2A receptor activation in the mPFC. For the behavioral studies, we did not selectively activate 5-HT2A receptors in the mPFC but assumed that mPFC 5-HT2A receptors were being activated by systemic administration of DOI since (1) phenethylamine hallucinogens induce head shakes when directly infused into the mPFC (Granhoff et al., 1992; Willins and Meltzer, 1997) and (2) systemic administration of a mGlu2/3 agonist LY354740 suppresses head shakes induced by either systemic (Gewirtz and Marek, 2000) or i. c.v administration of DOI (unpublished observations, G.J. Marek). Additionally, we examined the combined effects of a subanesthetic dose of MK-801 and DOI on head shakes and locomotor activity to test for additive or synergistic behavioral effects. Selective blockade of AMPA receptors suppressed 5-HTinduced EPSCs and DOI-induced head shakes/locomotor activity. A complementary synergistic effect of MK-801 on DOI-induced

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head shakes and locomotor activity was observed. These electrophysiological and behavioral effects further support a functional convergence of both psychotomimetic channel blocking NMDA receptor antagonists and serotonergic hallucinogens at enhancing glutamatergic neurotransmission which then results in stimulation of AMPA receptors in the mPFC. 2. Methods 2.1. Brain slice preparation Brain slices were prepared from male Sprague–Dawley rats (Harlan, Indianapolis, IN; 120–200 g) as described previously (Aghajanian and Rasmussen, 1989). Briefly, rats were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and decapitated. Coronal slices (500 μm) were cut with a oscillating-blade tissue slicer at a level corresponding to approximately 2.5 mm anterior to bregma (Paxinos and Watson, 1997). A slice containing the mPFC 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 34 °C with normal artificial cerebrospinal fluid (ACSF) which consisted of (in mM) NaCl 126; KCl 3; CaCl22;MgSO4 2; NaHCO3 26; NaH2PO4 1.25; D-glucose 10. 2.2. Electrophysiological recordings Intracellular recording and single-electrode voltage clamping were conducted in layer V pyramidal cells in the mPFC using an Axoclamp-2A (Axon Instruments Inc., Burlingame, CA) as previously described (Aghajanian and Marek, 1997). Stubby electrodes (∼ 8 mm, shank to tip) with relatively low capacitance and resistance (30–60 MΩ) 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 IPSCs. 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 IPSPs. In contrast, the 5-HT-induced EPSCs recorded with non-chloride containing electrodes (i.e., potassium acetate or gluconate) at holding potentials near ECl are completely blocked by the AMPA/kainate receptor antagonist LY293558 (Aghajanian and Marek, 1997). The voltage-clamp signals were low-pass filtered (1000 kHz) and data were acquired with a pCLAMP/Digidata 1200 system (Axon Instruments Inc.). EPSC frequencies were obtained from 10 successive episodes (one second duration) during the baseline and drug treatment periods. Evoked potentials were obtained while holding cells at − 80 mVand stimulating the forceps minor in the white matter deep to the cortex. 2.3. Electrophysiological data analysis EPSC frequency and amplitude were determined with a “Mini Analysis Program” (Synaptosoft, Inc.,www.synaptosoft.com); using thresholds of 5 pA and an area of ∼150 fC s− 1 for synaptic currents. Statistical comparisons of within-cell responses were

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made using two-tailed paired t-tests requiring p b 0.05 for statistical significance. The Kolmogorov–Smirnov test was used to determine if significant changes (p b 0.05) occurred in the EPSC amplitude or inter-event interval cumulative probability plots. The determination of EC50 values for the suppression of 5-HTinduced increases in EPSC frequency were calculated by nonlinear curve fitting (Graphpad Prism,www.Graphpad.com). 2.4. Behavioral experiments Forty-seven male Sprague–Dawley rats (180–280 g, Harlan, Indianapolis, IN) were housed in suspended stainless steel wire cages (18 × 36 × 20 cm) with 2–4 rats occupying each cage. The

colony room was maintained at ∼ 20 °C and relative humidity (60%). The room was illuminated 12 hr/day (07:00–19:00). All rats had free access to laboratory chow (Teklad 4% Rat Diet) and water except during experimental sessions. Most animals were injected with a low to moderate dose of DOI (0.313– 1.25 mg/kg, i.p.) multiple times but with at least one week between injections to minimize homologous receptor downregulation. The National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 80–23, revised 1996) was followed with respect to both the electrophysiological and behavioral experiments. All experiments were performed between 10:00 and 16:00. The animals were transferred to an individual clear polycarbonate

Fig. 1. The AMPA receptor antagonist LY300164 suppresses 5-HT-induced EPSCs in a concentration-dependent manner. a) Two consecutive and representative one second traces are shown for an intracellular recording from a layer V pyramidal cell from the rat mPFC under basal condition. In this cell, 5-HT increased the frequency of EPSCs by 35.7/s from a basal EPSC frequency of 2.4/s (b); LY300164 suppressed the frequency of 5-HT-induced EPSCs in a concentration-dependent manner (c–g; 0.3–30 μM).

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cage (43 × 21.5 × 20 cm) with a sawdust covered floor. All animals were habituated to this environment for 15 min before receiving a saline injection (1 ml/kg, i.p.). They were then observed for a 30 min period. One or two days later, the rats were reintroduced into this same environment for a 15 min habituation period. The animals were then injected with either vehicle or the AMPA/ kainate receptor antagonists LY293558 (3 mg/kg, i.p.) or GYKI 52466 (7.5 mg/kg, i.p.) or the NMDA receptor antagonist MK801 (0.2 mg/kg, i.p.). Then 15 min later (AMPA receptor antagonist experiments) or 30 min later (NMDA receptor antagonist experiments), the rats were injected with either saline or 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; i.p., 0.313, 0.63, 1.25 mg/kg). A within-subjects design was used for each of the glutamate receptor antagonist experiments with a single DOI dose. 2.5. Behavioral data analysis The animals were observed for 30 min following the DOI injection. The observation period began 1 min following the DOI injection to allow for drug absorption. The observer scored only one animal at a time. Head shake responses were counted in consecutive 5 min periods. Forward locomotion (movement from one end to the other end of the cage was scored as one cross) and rearing (only back two legs in contact with the ground) were also scored. All data are expressed as the mean ± S.E. The raw data were analyzed with a repeated measures ANOVA (Statistica,www.statsoft.com) and the significance level for multiple comparisons were corrected by using the Neuman–Keuls test (p ≤ 0.05). 2.6. Drugs The drugs used in this study were obtained from the following suppliers: Sigma (St. Louis, MO, USA: 5-HT creatine sulfate; D (−)-2-amino-5-phosphonopentoic acid (APV); α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA); 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466-HCl); (1-(2,5-dimethoxy-4-iodophenyl))-2-aminopropane HCl, (DOI)) and Alomone Labs (Jerusalem, Israel: tetrodotoxin (TTX)). (R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4,5-h) (2,3) benzodiazepine (LY300164 or Talampanel) and (3S,4aR,6R,8aR)-6[2-(1(2)Htetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid (LY293558) were kindly provided from the Lilly Research Laboratories by Dr. Darryle D. Schoepp (Indianapolis, IN).

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− 70.2 ± 1.1 mV; action potential amplitude, 82.4 ± 1.5 mV; action potential duration (at half amplitude), 0.75 ± 0.05 msec; input resistance (− 0.4 nA test pulse), 36.3 ± 2.7 MΩ (n = 47). All of the cells in the present series had the previously reported characteristics (Connors and Gutnick, 1990; McCormick et al., 1985) of regularly spiking pyramidal cells. 3.2. AMPA antagonists, but not a NMDA antagonist suppress 5-HT-induced EPSCs The 2,3-benzodiazepine AMPA receptor antagonist LY300164 blocked the 5-HT-induced EPSCs in a concentration-dependent manner with an IC50 = 1.4 ± 0.9 (n = 4, Figs. 1 and 2). Virtually complete blockade of the 5-HT-induced EPSCs was observed at the 30 μM concentration. This selective AMPA receptor antagonist decreased the amplitude of 5-HT-induced EPSCs at a concentration which blocked the frequency of 5-HT-induced EPSCs by ∼50% (n = 4, Kolmogorov–Smirnov test, p b 0.05). A decahydroisoquinoline AMPA/kainate (iGluK5) receptor antagonist LY293558 also suppressed the 5-HT-induced EPSCs in a concentration-dependent manner with an IC50 = 0.18± 0.04 μM; with virtually complete blockade at the 3 μM concentration (n = 4; Fig. 2). A drug which decreases the rate of AMPA receptor desensitization, cyclothiazide (100 μM × 20 min) increased the amplitude of the 5-HT-induced EPSCs in 2 of 3 cells examined in preliminary experiments (K–S test, p b 0.05, not shown) similar to previous results (Zhang and Marek, 2007). The competitive NMDA receptor antagonist APV (50 μM) decreased the frequency of 5-HT-induced EPSCs by only 6.4% (21.0 ± 5.2 EPSCs/s, control; 19.6 ± 4.9 EPSCs/s, APV; n = 8, not shown). In a subset of these same cells, APV completely blocked the frequency of NMDA-induced EPSCs (53.9 ± 15.2 EPSCs/s, control; − 3.4 ± 3.1 EPSCs/s, APV; n = 5, not shown). The specificity of the AMPA receptor antagonists in blocking post-synaptic AMPA receptors in layer V pyramidal cells was also assessed. These experiments were performed following treatment of the slices with 2 μM TTX to block fast sodium channels. Under

3. Results 3.1. Electrophysiological characteristics of layer V mPFC neurons Layer V pyramidal cells of the medial prefrontal cortex (predominantly in the pre-limbic area; Cg3) were recorded in a zone ca 1/2–2/3 the distance between the pial surface and the sub-cortical white matter. The pyramidal cells in the present study had the following characteristics: resting potential,

Fig. 2. AMPA and AMPA/iGluK5 receptor antagonists potently and completely suppress 5-HT-induced EPSCs in a concentration-dependent manner. LY300164 (n = 4) and LY293558 suppress the 5-HT-induced EPSCs with EC50's of 1.4 and 0.18 μM, respectively and complete suppression at 30 and 3 μM, respectively. The mean frequency of 5-HT-induced EPSCs over basal conditions were 23.7 ± 7.0 (LY300164) and 16.6 ± 7.2 (LY293558).

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examine whether DOI (1.25 mg/kg)-induced head shakes would be suppressed under conditions in which AMPA receptors would be blocked. GYKI 52466 blocked the increases in DOI-induced head shakes, forward locomotion (Fig. 3). The repeated measures ANOVA for head shakes found significant main effects of DOI (F(1,14) = 114.2, p b 0.001), GYKI 52466 (F(1,14) = 7.30, p b 0.05), and a significant DOI/ GYKI interaction (F(1,14) = 6.43, p b 0.05). The repeated measures ANOVA for horizontal locomotion found significant main effects of DOI (F(1,14) = 150.4, p b 0.001), GYKI 52466 (F(1,14) = 7.92, p b 0.05), and a significant DOI/GYKI interaction (F(1,14) = 6.08, p b 0.05). The effects of DOI and GYKI 52466 on rearing were similar to the effects on locomotor activity (not shown). Pre-treatment with GYKI 52466 (7.5 mg/ kg, 15 min prior to DOI) suppressed head shakes, hyperlocomotion, and rearing induced by DOI (1.25 mg/kg, p b 0.01, Newman–Keuls test). These results for DOI-induced head shakes with GYKI 52466 were confirmed with a second structurally distinct AMPA/

Fig. 3. The selective AMPA receptor antagonist GYKI 52466 attenuates the DOIinduced increase in head shakes and locomotor activity. A 7.5 mg/kg dose of the 2,3-benzodiazepine AMPA receptor (i.p., 15 min pre-treatment; n = 15) attenuated the increase in head shakes and cage crosses induced by DOI (1.25 mg/kg, i.p.) and recorded over a 30 min period. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 significantly greater than vehicle/vehicle; Dp b 0.05, DDp b 0.01 significantly less than DOI/vehicle; GG p b 0.01, GGGp b 0.001 significantly greater than vehicle/GYKI.

these conditions where AMPA receptors would be expected to be desensitized by bath application of AMPA, LY300164 (30 μM) blocked the AMPA (5 μM)-induced steady state inward current by 81 ± 3% (815 ± 498 pA, pre-antagonist inward current amplitude; not shown) while blocking the kainate (6 μM)-induced steady state inward current by only 7 ± 5% (652 ± 395 pA, pre-antagonist inward current amplitude) in the same four cells. Similarly, LY293558 (3 μM) blocked the AMPA (5 μM)-induced steady state inward current by 96 ± 2% (646 ± 300 pA pre-antagonist inward current; not shown) while blocking the kainate (6 μM)-induced steady state inward current by only 27 ± 16 % (401± 149 pA preantagonist inward current) in the same four cells. 3.3. AMPA receptor antagonists suppress DOI-induced head shakes The selective AMPA receptor antagonist GYKI 52466 (7.5 mg/kg, i.p.; n = 15), a 2,3-benzodiazepine, was used to

Fig. 4. The AMPA/kainate receptor antagonist LY293558 attenuates the DOIinduced increase in head shakes. A 3 mg/kg dose of the dihdroquinolinone AMPA receptor antagonist LY293558 (i.p., 15 min pre-treatment; n = 15) attenuated the increase in head shakes induced by DOI (1.25 mg/kg, i.p.) and recorded over a 30 min period. LY293558 did not alter the frequency of DOIinduced locomotor activity. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 significantly greater than vehicle/vehicle; DDp b 0.01 significantly less than DOI/vehicle.

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kainate receptor antagonist LY293558, a dihydroquinolinone (7.5 mg/kg, i.p., n = 15) which only potently blocks iGluK5 among the kainate receptor family. Again, LY293558 blocked the DOI-induced head shakes, though the AMPA/kainate receptor antagonist did not block the DOI-induced increase in forward locomotion or rearing (Fig. 4). The repeated measures ANOVA for head shakes found a significant main effects of DOI (F(1,14) = 91.4, p b 0.001), a trend for a significant main effect of LY293558 (F(1,14) = 3.30, p = 0.091), and a significant DOI/ LY293558 interaction (F(1,14) = 4.56, p b 0.05). The repeated measures ANOVA for horizontal locomotion found a significant main effects of DOI (F(1,14) = 9.30, p b 0.01) but non-significant effects of LY293558 (F(1,14) = 0.001) and a non-significant DOI/LY293558 interaction (F(1,14) = 0.17). The increase in rearing observed with DOI alone was not significantly decreased by LY293558 (not shown). Pre-treatment with LY293558 (3 mg/ kg, 15 min prior to DOI) blocked DOI (1.25 mg/kg)-induced head shakes by 29 % (p b 0.05, Fig. 4).

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3.4. A non-competitive NMDA receptor antagonist potentiates DOI-induced head shakes The noncompetitive NMDA receptor antagonist MK-801 was used to examine whether DOI-induced head shakes would be modulated by blockade of NMDA receptors. The first experiment examined a low dose of DOI (0.313 mg/kg) that does not significantly induce an increased frequency of head shakes (Fig. 5). The ANOVA on headshake frequency found a significant DOI effect (F(1,12) = 4.55, p b 0.05), a significant MK-801 effect (F(1,12) = 17.60, p b 0.01), and a significant interaction of the drugs (F(1,12)= 4.34, p b 0.05). The frequency of DOI-induced head shakes in the MK-801 treated rats was significantly greater than in the vehicle/vehicle condition (p b 0.01, Newman–Keuls test), the vehicle/MK-801 condition (p b 0.01), and the DOI/ vehicle condition (p b 0.05). In these same rats, we also observed a significant main effect of MK-801 on cage crosses (F(1,12) = 15.43, p b 0.005). The frequency of cage crosses in the DOI/MK-

Fig. 5. Complementary synergistic effects occurred for the combination of DOI and the channel blocking NMDA receptor antagonist MK-801. The first row of graphs shows the synergistic effects of a low dose of DOI (0.313 mg/kg, i.p.) and MK-801 (0.2 mg/kg, i.p., 30 min pre-treatment prior to DOI; n = 13) from the first experiment on head shakes and locomotor activity. The second row of graphs shows the synergistic effects of a medium DOI dose (0.625 mg/kg, i.p.) and MK-801 (0.2 mg/kg, i.p.; n = 10) from the second experiment on head shakes and locomotor activity. The third row of graphs shows the effects of a high DOI dose (1.25 mg/kg, i.p.) and MK-801 (0.2 mg/kg, i.p.; n = 20) from the third experiment on head shakes and locomotor activity. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 significantly greater than vehicle/vehicle; Dp b 0.05, DDp b 0.01, DDD p b 0.001 significantly greater than DOI/vehicle; Mp b 0.05, MMp b 0.01, MMMp b 0.001 significantly greater than vehicle/MK-801.

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801 treated rats was significantly greater than in the vehicle/ vehicle condition (p b 0.05), the vehicle/MK-801 condition (p b 0.05), and the DOI/vehicle condition (p b 0.01). The second experiment examined a moderate dose of DOI (0.625 mg/kg) that does significantly increase DOI-induced head shakes. The headshake ANOVA found a significant DOI effect (F(1,9) = 36.82, p b 0.001), a non-significant MK-801 effect (F(1,9) = 3.16, p = 0.109), and a trend for the interaction of the drugs (F(1,9) = 3.35, p = 0.10; Fig. 5). The frequency of DOI-induced head shakes in the MK-801 treated rats was significantly greater (Newman–Keuls test) than in the vehicle/ vehicle condition (p b 0.001), the vehicle/MK-801 condition (p b 0.001), and the DOI/vehicle condition (p b 0.05). In these same rats, we also observed a significant main effect of MK-801 on cage crosses (F(1,9) = 7.29, p b 0.05) and a significant main effect of DOI on cage crosses (F(1,9) = 6.11, p b 0.05). The frequency of cage crosses in the DOI/MK-801 treated rats was significantly greater than in the vehicle/vehicle condition (p b 0.05), and the DOI/vehicle condition (p b 0.05). This experiment involving a middle dose of DOI (0.625 mg/kg) and MK-801 was the only one where the combination of DOI and MK-801 resulted in significantly more rearing than in either drug condition alone (not shown). The third DOI/MK-801 experiment involved a near-maximal dose of DOI (1.25 mg/kg) with respect to increasing head shake frequency. The head shake ANOVA revealed only a significant effect of DOI (F(1,19) = 79.0, p b 0.001) without significant effects of either MK-801 (F(1,19) = 0.48) or the interaction of DOI and MK-801 (F(1,19) = 0.49; Fig. 5). Significant increases in head shake frequency were observed only for the DOI/vehicle group and the DOI/MK-801 group (p b 0.001). However, it should be noted that in these same rats, we observed a significant effect on locomotor activity for MK-801 (F(1,19) = 28.4, p b 0.001), DOI (F(1,19) = 23.6, p b 0.001) and the DOI/MK801 interaction (F(1,19) = 17.8, p b 0.001). The vehicle/MK-801 group and the DOI/MK-801 group were significantly greater than the vehicle/vehicle group (p b 0.01 and 0.001, respectively). The vehicle/MK-801 and DOI/MK-801 group were also significantly greater than the DOI/vehicle group (p b 0.05 and p b 0.001, respectively). Finally, the DOI/MK-801 group was significantly greater than the vehicle/MK-801 group. 4. Discussion The two main findings from the present studies is that (1) activation of AMPA receptors appears to mediate the 5-HT-induced increase in EPSC frequency recorded from layer V pyramidal cells of the mPFC and (2) the DOI-induced head shake response in rats appears to be mediated, in part, via activation of AMPA receptors. Previously, the modulation by non-NMDA receptors of excitatory synaptic currents/potentials induced by 5-HT2A receptor activation could not be differentiated between AMPA vs. kainate receptor mediation based on the receptor selectivity of the AMPA/kainate antagonist employed (Aghajanian and Marek, 1997; Marek and Aghajanian, 1999). LY293558 is an AMPA receptor antagonist which also potently blocks the iGluK5 receptor subtype (Bleakman et al., 1996; Schoepp et al., 1995), leaving unresolved whether

heterodimeric kainate receptors containing the iGluK5 receptor subunit are involved in this effect (Bleakman et al., 1999). LY300164 and GYKI 52466 are selective 2,3-benzodiazepine AMPA receptor antagonists employed in the electrophysiological and behavioral experiments, respectively (Bleakman et al., 1996; Cotton and Partin, 2000). Both LY293558 and a selective AMPA receptor antagonist suppressed the 5-HT-induced EPSCs and the DOI-induced head shakes. Previous work has found that the DOIinduced head shakes are supported by local hallucinogenic drug administration in the mPFC (Willins and Meltzer, 1997). Therefore, these studies taken together with previous in vivo results suggesting important modulatory effects by mGlu2 receptors (Gewirtz and Marek, 2000; Klodzinska et al., 2002; Marek et al., 2000; Zhai et al., 2003; Benneyworth et al., 2007) are consistent with the hypothesis that stimulation of AMPA receptors mediates physiologically important effects of 5-HT2A receptor activation in the mPFC (Pei et al., 2004; Scruggs et al., 2000a). Local application of AMPA receptor antagonists onto mPFC slice preparations was able to completely block 5-HT-induced EPSCs recorded from layer V pyramidal cells. In contrast, only 58% and 40% decreases in DOI-induced head shakes were observed after the AMPA/kainate receptor antagonist and the selective AMPA receptor antagonist, respectively. The doses chosen for LY293558 (3 mg/kg, i.p.) and GYKI 52466 (7.5 mg/ kg, i.p.) were used in order to stay below doses that might induce ataxia or other non-specific motor disruption (Simmons et al., 1998). The dose of GYKI 52466 employed (7.5 mg/kg, i.p.) was also chosen on the basis that 7.5–25 mg/kg doses of GYKI 52466 were able to block DOI-induced changes in c-fos expression or Arc mRNA expression in the mPFC (Scruggs et al., 2000; Pei et al., 2004). The combination of the slice electrophysiology experiments together with the behavioral experiments suggests that the DOI-induced head shake response is mediated by either (1) indirect AMPA receptor stimulation following cortical 5-HT2A receptor activation or (2) a combination of a direct effect via stimulation of cortical 5-HT2A receptors on layer V mPFC pyramidal cells plus a simultaneous indirect glutamatergic modulation following 5-HT2A receptor activation or (3) independent modulation of cortical layer V pyramidal cells by AMPA receptor stimulation (Puig et al., 2003) and some combination of direct/indirect effects of 5-HT2A receptor activation. The failure of the AMPA receptor antagonists to completely block the DOI-induced head shakes also could be due to a sub-maximal dose employed or opposing influences of AMPA receptor blockade in other brain regions. While 5-HT2A receptors in the mPFC and neocortex are located in parvalbuminexpressing GABAergic interneurons, glia and to a much lesser extent in axons, the robust expression of 5-HT2A receptors in pyramidal neurons would make it likely that either of the latter two scenarios explains the relatively modest effect of AMPA receptor antagonists on DOI-induced head shakes (CorneaHebert et al., 1999; Jakab and Goldman-Rakic, 2000; Miner et al., 2003; Willins et al., 1997; Xu and Pandey, 2000). However, the heterogenous compartmentalization of 5-HT2A receptors in the pre-frontal cortex suggests caution in extrapolating mechanisms underlying any given in vitro finding to a range of in vivo effects induced by phenethylamine hallucinogens.

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The enhancement of DOI-induced head shakes by the NMDA channel blocker MK-801 is consistent with previous behavioral studies. In mice, NMDA was found to suppress 5-HT-induced head twitches while NMDA receptor antagonists enhanced head twitches mediated by 5-HT2A receptor activation (Kim et al., 1998; Kim et al., 1999). The channel blocking non-competitive NMDA receptor antagonist MK-801 has also been previously shown to enhance DOI-induced head shakes and locomotion (Dall'Olio et al., 1999) in rats. At first glance, enhancement of DOI-induced head shakes by NMDA receptor antagonists appears paradoxical when compared to the suppression of DOIinduced head shakes by AMPA receptor antagonists. However, systemic administration of either non-competitive NMDA receptor antagonists or a phenethylamine hallucinogen activating 5-HT2A receptors have been demonstrated to increase extracellular levels of glutamate in the pre-frontal cortex (Lopez-Gil et al., in press; Lorrain et al., 2003; Moghaddam et al., 1997; Moghaddam and Adams, 1998; Muschamp et al., 2004). Systemic or local (mPFC) administration of the 5-HT2A receptor antagonist M100907 has also been shown to block increased locomotor activity or motor impulsivity induced by either systemic or local (mPFC) infusions of NMDA receptor antagonists (Carli et al., 2004; Gleason and Shannon, 1997; Higgins et al., 2003; Martin et al., 1997). Furthermore, recent evidence with positive allosteric modulators of mGlu2 receptors suggest that most of the effects of mGlu2/3 receptor agonists on DOI-induced head shakes and NMDA receptor antagonistinduced locomotor hyperactivity is due to activation of mGlu2 autoreceptors (Benneyworth et al., 2006; Benvenga et al., 2006; Galici et al., 2005; Johnson et al., 2005). Furthermore and congruent with animal studies, acute administration of serotonergic hallucinogens or ketamine are both known to result in hyperfrontal metabolic patterns in healthy subjects (Vollenweider et al., 1997a; Vollenweider et al., 1997b). Thus, the synergistic effects of DOI and MK-801 on head shakes and locomotor activity are congruent with evidence for functional convergence of these two different psychotomimetic drug classes at increasing glutamatergic tone in the mPFC. The experiment involving DOI and MK-801 also points out differences in action that appear to result in complementary behavioral effects. MK-801 does not induce head shakes, unlike DOI, but does synergistically increase the frequency of DOIinduced head shakes. Conversely, DOI does synergistically increase the hyperlocomotor response to MK-801. Another potential difference between activation of 5-HT2A receptors and blockade of NMDA receptors is that thalamic afferents play a major role in 5-HT-induced EPSCs and hallucinogen-induced c-fos expression in the mPFC while hippocampal afferents appear to lead to excitation of mPFC pyramidal cells after systemic administration of NMDA receptor antagonists (Jodo et al., 2004; Lopez-Gil et al., in press; Marek et al., 2001; Scruggs et al., 2000). In another difference with channel blocking NMDA antagonists, an excitatory influence of 5-HT2A receptors on cortical pyramidal cells or potentially of glutamate release from afferents originating the claustrum appears to support 5-HT-induced EPSCs, DOI-induced head shakes and increases in immediate early gene activation (Gonzalez-Maeso et al., 2007; Weisstaub et al., 2006). Finally,

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somewhat different subjective effects have also been reported in healthy human subjects between serotonergic hallucinogens and the channel blocking NMDA receptor antagonists. The indoleamine hallucinogen N,N-dimethyltryptamine tended to have a stronger effect at inducing positive symptoms compared to ketamine. Conversely, ketamine tended to have a stronger effect on negative symptoms and catalepsy than N,N-dimethyltryptamine (Gouzoulis-Mayfrank et al., 2005). The present behavioral interactions between ionotropic glutamate receptor antagonists and DOI suggesting a functional convergence on glutamatergic pathways is also consistent with evidence suggesting glutamatergic modulation of 5-HT2A receptors. First, mGlu2/3 receptor agonists and antagonists modulate 5-HT-induced EPSCs recorded from mPFC layer V pyramidal cells in a fashion consistent with an autoreceptor function for mGlu2/3 receptors (Marek et al., 2000). Second, a laminar overlap exists for 5-HT2A receptors and mGlu2 receptors and glutamatergic afferents to the pre-frontal cortex arising from the midline and thalamic nuclei (Berendse and Groenewegen, 1991; Marek et al., 2000). Third, thalamic lesions decreased the frequency of 5-HT-induced EPSCs recorded from mPFC pyramidal cells by ∼ 60% (Lambe and Aghajanian, 2001; Marek et al., 2001). Fourth, chemical lesions of the medial thalamus producing near complete loss of glutamatergic thalamocortical afferents arising from the intralaminar and midline thalamic nuclei resulted in an increased binding of [125 I]DOI to pre-frontal cortical 5-HT2A receptors (Marek et al., 2001). Fifth, administration of the mGlu2/3 receptor agonist LY354740 on day two of a three day sub-chronic regimen of the 5-HT2A receptor agonist DOI prevented the down-regulation of [125I] DOI binding in the mPFC (Marek et al., 2006). These observations again highlight a number of diverse findings suggesting the interdependence of the serotonergic and glutamatergic systems with respect to 5-HT2A receptor activation and regulation. The present delineation of AMPA receptors in mediating important effects of cortical 5-HT2A receptor activation in electrophysiology or behavior add to previous work suggesting an autoreceptor function for mGlu2 receptors for both the electrophysiological and behavioral effects (Marek et al., 2000; Gewirtz and Marek, 2000; Benneyworth et al., 2007). Thus, the recent clinical report that a mGlu2/3 receptor agonist prodrug is an effective antipsychotic drug in schizophrenic patients (Schoepp, 2006; Patil et al., in press) is not surprising given (1) the preclinical observations that mGlu2/3 receptor agonists block a variety of electrophysiological, neurochemical and behavioral effects of either phenethylamine hallucinogens or channel blocking NMDA receptor antagonists and (2) the growing evidence for functional relationships between these different classes of psychotomimetic drugs involving glutamate and 5HT2A receptors. 5. Conclusion Both the mPFC slice electrophysiological experiments and the behavioral experiments with DOI and AMPA or NMDA receptor antagonists suggest that at least a portion of the behaviorally salient

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