QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.107490v1 320/1/437    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zhang, C. Articles by Marek, G. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, C. Articles by Marek, G. J.

NEUROPHARMACOLOGY

Group III Metabotropic Glutamate Receptor Agonists Selectively Suppress Excitatory Synaptic Currents in the Rat Prefrontal Cortex Induced by 5-Hydroxytryptamine_2A Receptor Activation

Department of Psychiatry, Yale University School of Medicine, Ribicoff Research Facilities of the Connecticut Mental Health Center, New Haven, Connecticut

Received May 6, 2006; accepted October 3, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Activation and blockade of prefrontal cortical 5-hydroxytryptamine_2A (5-HT_2A) receptors have been linked to the action of hallucinogenic and antidepressant/antipsychotic drugs; these effects may involve modulation of glutamate release from thalamocortical afferents. Although activation of metabotropic glutamate 2 (mGlu2) receptors may suppress 5-HT-induced excitatory postsynaptic currents (EPSCs), group III mGlu receptors (mGlu4/7/8) also are expressed in the thalamus and may suppress 5-HT-induced EPSCs. We have found by intracellular recordings from layer V pyramidal cells of the medial prefrontal cortex (mPFC) that group III mGlu receptor agonists (R,S)-4-phosphonophenylglycine (PPG), L-4-phosphono-2-aminobutyric acid (L-AP4), L-serine-O-phosphate (L-SOP), and (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) preferentially suppress 5-HT-induced EPSCs compared with excitatory postsynaptic potentials evoked by electrical stimulation of the white matter. A number of pharmacological features [e.g., the rank order of agonist potency; MAP4 partial agonist action; differential potency for the group III mGlu receptor antagonist (R,S)--cyclopropyl-4-phosphonophenylglycine (CPPG) in blocking the suppressant action of PPG or MAP4; and a relatively low potency of 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid (LY341495) in blocking the suppressant action of PPG or L-SOP] suggest that activation of both mGlu4 and mGlu8 receptors may play a role in suppressing 5-HT-induced EPSCs. Furthermore, L-SOP did not alter the synaptic currents or steady-state inward current induced by -amino-3-hydroxy-5-methylisoxazole-4-proprionic acid. Thus, although both group III and group II mGlu receptor agonists suppress the frequency of 5-HT-induced EPSCs in the mPFC, they differ in that the group III mGlu receptor agonists appear to have relatively minimal effects on glutamate released by sources other than thalamocortical afferents.

We have previously found by recording from layer V pyramidal cells in the medial prefrontal cortex (mPFC) that activation of µ-opioid and metabotropic glutamate 2 (mGlu2) receptors suppresses 5-HT-induced excitatory postsynaptic currents (EPSCs) that are mediated by 5-HT_2A receptor activation (Aghajanian and Marek, 1997; Marek and Aghajanian, 1998, 1999; Marek et al., 2000). The mRNA for both µ-opioid and mGlu2 receptors is especially abundant in the midline and intralaminar nuclei of the thalamus (Ohishi et al., 1993; Mansour et al., 1994). Both group II mGlu (mGlu2/3) and group III mGlu (mGlu4/6/7/8) receptors are known to suppress transmitter release via presynaptic action. In a number of locations throughout the brain, members of both the group II and III mGlu receptors appear to be present within the same terminals (Shigemoto et al., 1997). In this respect, mGlu4 and mGlu7 receptor mRNA is relatively strongly expressed within the midline and intralaminar thalamic nuclei (Ohishi et al., 1995). The mRNA for the mGlu8 receptor is also reported to be present in the thalamus (Saugstad et al., 1997). Therefore, the purpose of the present studies was to determine whether a member(s) of the group III mGlu receptors suppresses 5-HT-induced EPSCs while recording from layer V pyramidal cells of the medial prefrontal cortex. We also tested the specificity of group III mGlu receptor agonists in suppressing 5-HT-induced EPSCs compared with other modes of relatively nonspecific release of excitatory amino acids, such as electrical stimulation of the white matter deep to the cortex and bath application of the prefrontal slice with the ionotropic glutamate agonist amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA). We found that 1) the group III mGlu agonists selectively suppressed 5-HT-induced EPSCs compared with other indices of excitatory transmission; and (2) the evidence is consistent with the working hypothesis that activation of mGlu4 and mGlu8 receptors mediates the suppressant action of group III mGlu receptor agonists on 5-HT-induced EPSCs.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Electrophysiology. Brain slices were prepared from male Sprague-Dawley rats (weight 120–200 g) (Harlan, Indianapolis, IN) as described previously (Aghajanian and Marek, 1997). In brief, rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and decapitated. Coronal slices (500 µm) were cut with an oscillating blade tissue slicer at a level corresponding to approximately 2.5 mm anterior to bregma. 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, 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 using an Axoclamp-2A (Axon Instruments, Burlingame, CA) as described previously (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%) is blocked by bicuculline during intracellular recordings using KCl-containing electrodes, suggesting the presence of some reverse inhibitory postsynaptic potentials. In contrast, the 5-HT-induced EPSCs recorded with nonchloride-containing electrodes (i.e., potassium acetate or gluconate) at holding potentials near E_Cl are completely blocked by the AMPA/kainate 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). 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 to the cortex.

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 for synaptic currents. Statistical comparisons of within-cell responses were made using two-tailed paired t tests requiring p < 0.05 for statistical significance. The Kolmogorov-Smirnov (K-S) test was used to determine whether significant changes (p < 0.001) occurred in the EPSC amplitude or interevent interval cumulative probability plots. The determination of EC₅₀ values for the suppression of 5-HT-induced increases in EPSC frequency or of evoked excitatory postsynaptic potentials (EPSPs) was calculated by nonlinear curve fitting (Prism; GraphPad Software Inc., San Diego, CA).

We have previously found that group II mGlu receptor agonists selectively suppress the frequency of 5-HT-induced EPSC relative to the amplitude of these synaptic currents (Marek et al., 2000). In contrast, the AMPA receptor antagonist LY 300164 did significantly decrease the amplitude of 5-HT-induced EPSCs at a concentration of the antagonist near the EC₅₀ for suppression of the frequency of 5-HT-induced EPSCs in all of the five cells tested (G. Marek and C. Zhang, unpublished observations). In contrast to the group II mGlu receptor agonists, the group II mGlu receptor antagonist LY341495 clearly increased the frequency of 5-HT-induced EPSCs (consistent with the presence of mGlu2 receptors in thalamocortical afferents) when using "sharp" electrodes under voltage-clamp conditions. Analysis of EPSCs recorded under these conditions allows for expected pharmacological modulation of these synaptic currents (e.g., Schild analysis) when considering 5-HT_2A, µ-opioid, and mGlu2 receptor pharmacology (Marek and Aghajanian, 1998, 1999; Marek et al., 2000). Furthermore, similar effects (5-HT agonist concentration-response relationships; similar degree of suppression of 5-HT-induced EPSCs by thalamic lesions) have been obtained comparing results from experiments using "sharp" electrodes versus whole-cell recording conditions (Aghajanian and Marek, 1997; Lambe and Aghajanian, 2001; Marek et al., 2001).

Compounds. The drugs used in this study were obtained from the following suppliers: 5-HT creatine sulfate (Sigma, St. Louis, MO); AMPA (RBI, Natick, MA); L-(+)-2-amino-4-phophonobutyric acid (L-AP4), O-phospho-L-serine or L-serine-O-phosphate (L-SOP), (R,S)-4-phosphonophenylglycine (PPG), (R,S)--cyclopropyl-4-phosphonophenylglycine (CPPG), (S)-2-amino-2-methyl-4-phosphonbutanoic acid (MAP4), and (2S)--ethylglutamic acid (EGLU) (Tocris, Ballwin, MO); and tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel). LY354740 and LY341495 were provided by Drs. James A. Monn and Darryle D. Schoepp of the Lilly Research Laboratories (Indianapolis, IN).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Layer V pyramidal cells of the medial prefrontal cortex (predominantly in the prelimbic area; Cg3) were recorded in a zone approximately one-half to two-thirds the distance between the pial surface and the subcortical white matter. The pyramidal cells in the present study had the following characteristics: resting potential, –71.0 ± 0.7 mV; action potential amplitude, 82.1 ± 0.7 mV; action potential duration (at half-amplitude), 0.79 ± 0.02 ms; and input resistance (–0.4 nA test pulse), 34.9 ± 1.7 M (n = 142). All of the cells in the present series had the previously reported characteristics of regularly spiking pyramidal cells (McCormick et al., 1985).

Group III mGlu Agonists Selectively Suppress 5-HT-Induced EPSCs. Agonists selective for group III relative to group II mGlu receptors (Schoepp et al., 1999) potently suppressed 5-HT-induced EPSCs. L-SOP (3–1000 µM) maximally suppressed the frequency of 5-HT-induced EPSCs by 70.0% and with an EC₅₀ = 19.9 ± 2.1 µM (Figs. 1 and 2). In all of the five cells tested with L-SOP, 5-HT increased the EPSC amplitude (K-S test, p < 0.01) compared with the basal EPSC amplitude. In only two of these five cells did a concentration of L-SOP, which decreased the frequency of 5-HT-induced EPSCs by 50%, also decrease the amplitude of 5-HT-induced EPSCs (K-S test, p < 0.01). In none of the five cells did L-SOP decrease either the amplitude or frequency of basal EPSCs.

View larger version (36K): [in this window] [in a new window]   Fig. 1. The group III mGlu receptor agonist L-SOP suppresses 5-HT-induced EPSCs at concentrations that do not appreciably reduce electrically evoked EPSPs. A, three consecutive and representative 1-s traces are shown for an intracellular recording from a layer V pyramidal cell from the rat mPFC. In this cell, 5-HT increased the frequency of EPSCs by 28.1/s from a basal EPSC frequency of 4.5/s (A2); L-SOP suppressed the frequency of 5-HT-induced EPSCs in a concentration-dependent manner (A3–A6). B, in a different layer V pyramidal cell from the mPFC, there was only a minimal (10%) suppression of the electrically evoked EPSP when stimulating the white matter deep to the cortex. The basal electrically evoked EPSPs averaged 15.2 mV.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Group III mGlu receptor agonists preferentially suppressed 5-HT-induced EPSCs compared with electrically evoked EPSP. Top, PPG (n = 5), L-AP4 (n = 6), and L-SOP (n = 5) suppress the 5-HT-induced EPSCs with EC50 of 0.37, 8.9, and 19.9 µM, respectively, and maximal suppression of 70 to 96%. MAP4 (n = 5) suppressed 5-HT-induced EPSCs by 38% with an EC50 of 58 µM. The mean frequency of 5-HT-induced EPSCs over basal conditions were 35.2 ± 6.2 (PPG), 24.7 ± 4.1 (L-AP4), 31.7 ± 5.3 (L-SOP), and 18.5 ± 2.9 EPSC/s (MAP4). In contrast (bottom), PPG (n = 5) and L-AP4 (n = 6) suppressed electrically evoked EPSPs by only 34 and 35.5% at the maximal concentrations tested, which suppressed 5-HT-induced EPSCs by 96 and 80%, respectively. L-SOP (n = 5) suppressed electrically evoked EPSPs by less than 10%. The mean amplitude of electrically evoked EPSPs was 14.7 ± 0.5 (PPG), 14.3 ± 3.0 (L-AP4), and 13.4 ± 0.8 mV (L-SOP).

View larger version (17K): [in this window] [in a new window]   Fig. 3. The group II mGlu receptor agonist (1S,3S)ACPD suppresses both 5-HT-induced EPSCs and electrically evoked EPSPs. (1S,3S)ACPD potently suppressed both 5-HT-induced EPSCs and electrically evoked EPSPs with EC50 of 17.1 and 141 µM, respectively, and maximal effects of 71 and 82%, respectively (n = 5). 5-HT (100 µM) induced 21 ± 5.7 EPSC/s over basal conditions, and the baseline electrically evoked EPSP amplitude was 16.1 ± 1.5 mV.

PPG (0.05–50 µM) maximally suppressed the frequency of 5-HT-induced EPSCs by 96% and with an EC₅₀ = 0.37 ± 0.10 µM (n = 5; Fig. 2). Application of PPG to the prefrontal slices resulted in a relatively shallow slope with a Hill coefficient of 0.59 ± 0.07. In all of the five cells tested with PPG, 5-HT increased the EPSC amplitude (K-S test, p < 0.001). In only three of these five cells did a concentration of PPG, which suppressed the frequency of 5-HT-induced EPSCs in each cell by 50% (p < 0.001), decrease the amplitude of 5-HT-induced EPSCs (K-S test, p < 0.01).

We also tested another commonly studied mGlu compound, MAP4 (30–300 µM, n = 5), which has been characterized as an antagonist at group III mGlu receptors but has also been reported to act as a partial agonist at the human mGlu4 receptor (Knopfel et al., 1995). The maximal suppression of 5-HT-induced EPSCs by MAP4 was only 38%, and the extrapolated EC₅₀ (using the 300 µM response as a maximal effect) was 58.0 µM (Fig. 2).

The ability of the group III mGlu receptor agonists to suppress electrically evoked EPSPs in layer V pyramidal cells was also tested. A major component of these electrically evoked EPSPs is probably caused by activation of descending corticofugal fibers originating from layers V and VI (Marek et al., 2000) because descending corticothalamic axons greatly outnumber ascending thalamocortical axons in most corticothalamic pathways. L-SOP (10–1000 µM) caused virtually no suppression of electrically evoked EPSPs (<10%, n = 5; Figs. 1 and 2). In contrast, L-AP4 (10–300 µM) did suppress the electrically evoked EPSCs, but only with a maximal suppression of 35.5% compared with the 80.1% suppression of 5-HT-induced EPSCs (n = 6; Fig. 2). The EC₅₀ for L-AP4 at suppressing the electrically evoked EPSPs was 18.9 ± 4.6 µM. PPG (0.05–50 µM) suppressed the electrically evoked EPSPs recorded from layer V pyramidal cells by only 34% at the maximal concentration tested (50 µM) and with an estimated EC₅₀ of 8.7 µM (n = 5; Fig. 2). Thus, PPG was at least 24-fold less potent at suppressing the electrically evoked EPSPs compared with the 5-HT-induced EPSCs.

A Group II mGlu Receptor Agonist: Similar Efficacy for Suppression of 5-HT-Induced EPSCs and Electrically Evoked EPSPs. Given previous potent and highly efficacious (>70% suppression) effects of group II mGlu receptor agonists on both 5-HT-induced EPSCs and electrically evoked EPSPs, the effects of the mGlu2/3 receptor agonist 1S,3S-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3S)ACPD] (10–300 µM), which also has weak activity at group I mGlu receptors, was tested for comparison with the group III mGlu receptor agonists. (1S,3S)ACPD potently suppressed 5-HT-induced EPSCs with an EC₅₀ of 17.1 µM and a maximal suppression of 71% (n = 5; Fig. 3). (1S,3S)ACPD also suppressed electrically evoked EPSPs also recorded from layer V pyramidal cells with an EC₅₀ of 141 µM and a maximal suppression of 82.4% (n = 5).

Group III mGlu Receptor Agonists and AMPA-Induced Responses. The ability of the selective group III mGlu receptor agonist L-SOP to suppress AMPA-induced synaptic currents and steady-state inward currents in layer V pyramidal cells of the medial prefrontal cortex was also compared with the highly selective mGlu2/3 receptor agonist LY354740. AMPA induced a robust increase in both the frequency and amplitude of EPSCs (p < 0.01; Fig. 4). A concentration of L-SOP (300 µM), which exerted near maximal efficacy in decreasing the frequency of 5-HT-induced EPSCs, did not alter the frequency of basal EPSCs (Figs. 4 and 5). Furthermore, L-SOP did not alter either the frequency or amplitude of AMPA-induced EPSCs. Testing of the individual cells with the K-S test revealed that L-SOP increased the interevent interval (e.g., decreased the frequency) for AMPA-induced EPSCs in only one of seven cells, whereas L-SOP decreased the amplitude of AMPA-induced EPSCs for each subsequent substance in only two of seven cells (p < 0.001). In none of these cells did L-SOP alter the basal EPSC interevent interval or amplitude. In this series of seven cells, L-SOP (300 µM) induced only small outward currents (38.3 ± 14.4 pA, mean ± S.E.M.; only one cell had a current >40 pA). Similar to the relative lack of efficacy on suppressing AMPA-induced EPSCs, L-SOP did not alter the AMPA-induced steady-state inward current (710 ± 89.6 pA, AMPA; 689 ± 148 pA, L-SOP/AMPA; Fig. 5).

View larger version (48K): [in this window] [in a new window]   Fig. 4. Differential effect of the mGlu2/3 receptor agonist LY354740 and the group III mGlu receptor agonist L-SOP on AMPA-induced EPSC. Compared with the basal state (A), bath application of AMPA (5 µM; B) induced a vigorous increase in the frequency of EPSCs as shown from five consecutive 1-s traces obtained when recording from a layer V pyramidal cell. L-SOP (300 µM; C), at a concentration that robustly suppressed 5-HT-induced EPSCs, did not appreciably alter the frequency or amplitude of AMPA-induced EPSCs (n = 6). In contrast, LY354740 (1 µM; D) decreased the frequency of AMPA-induced EPSCs (n = 6).

View larger version (18K): [in this window] [in a new window]   Fig. 5. L-SOP (300 µM x 3 min) does not alter the frequency or amplitude of AMPA-induced EPSC or the AMPA-induced inward current when recording from layer V pyramidal cells of the mPFC. Summary (top) graph showing the robust increase in the frequency of AMPA-induced EPSCs and a lack of effect for the group III mGlu receptor agonist L-SOP on this measure (n = 7). The middle summary graph similarly shows a lack of effect of L-SOP on the amplitude of AMPA-induced EPSC. The lower summary graph shows that L-SOP does not alter the AMPA-induced steady-state inward current (n = 7). A = AMPA (5 µM x 40 s). #, p < 0.05 and ##, p < 0.01 comparing AMPA alone or AMPA/L-SOP with basal or L-SOP alone, respectively. *, p < 0.05 comparing AMPA/L-SOP with AMPA alone for primary a priori t test.

The ability of the highly selective group II mGlu agonist LY354740 to suppress responses induced by bath application of AMPA was tested in five of the same cells in which L-SOP was tested. LY354740 (1 µM, total n = 6) did not alter the basal EPSC frequency or amplitude, consistent with previous observations (Marek et al., 2000). LY354740 did suppress the frequency of AMPA-induced EPSCs (p < 0.05) by 60 ± 8% (Figs. 4 and 6). Testing of the individual cells with the K-S test revealed that LY354740 decreased the AMPA-induced EPSC frequency in five of six cells, whereas LY354740 decreased the amplitude of AMPA-induced EPSCs in only two of six cells (p < 0.01). In none of these cells did LY354740 alter the basal EPSC interevent interval or amplitude. In this series of six cells, LY354740 induced an average outward current of 70 ± 35 pA (three of six cells exhibited outward currents of 90 pA). LY354740 also decreased the AMPA-induced steady-state inward current by 48% (p < 0.01). Interestingly, the preliminary observations found that the EC₅₀ of LY354740 for suppression of AMPA-induced EPSCs was 4–13-fold higher (375 and 1150 nM, n = 2) than we have previously observed for suppression of 5-HT-induced EPSCs (EC₅₀ = 89 nM) (Marek et al., 2000).

View larger version (18K): [in this window] [in a new window]   Fig. 6. The mGlu2/3 receptor agonist LY354740 decreases the frequency of AMPA-induced EPSC and decreases the amplitude of the AMPA-induced inward current when recording from layer V pyramidal cells of the mPFC. Summary (top) graph showing the robust increase in the frequency of AMPA-induced EPSCs and a significant effect for LY354740 on this measure (p < 0.05; n = 6). The middle summary graph similarly shows a lack of effect of LY354740 on the amplitude of AMPA-induced EPSCs. The lower summary graph shows that LY354740 decreases the amplitude of the AMPA-induced steady-state inward current (p < 0.05; n = 5). A = AMPA (5 µM x 40 s), LY354 = LY354740 (300 nM x 5 min). *, p < 0.05 and **, p < 0.01 comparing AMPA/LY354740 with AMPA alone for an a priori t test of interest. #, p < 0.05 and ##, p < 0.01 comparing AMPA alone or AMPA/LY354740 with basal or LY354740 alone, respectively.

mGlu Receptor Antagonists Block the Suppressant Action of Group III Agonists. We examined the potency of antagonists in blocking the suppressant action of group III mGlu receptor agonists with two different strategies. The first was to compare the potency of the group III mGlu receptor antagonist CPPG in suppressing the effects of PPG or MAP4 with a full Schild analysis. The second strategy was to take advantage of the differing potency of the competitive mGlu receptor antagonist LY341495 in blocking the group III mGlu receptors so that the group III mGlu receptor pharmacology would be determined in the presence of the mGlu2/8 autoreceptor blockade.

PPG, in the absence of mGlu receptor antagonists, exhibited a relatively shallow curve for the suppression of 5-HT-induced EPSCs with an EC₅₀ of 0.37 µM. CPPG (10–100 µM; Fig. 7) resulted in a rightward shift of the concentration-response curve for PPG-induced suppression. The pA₂ for CPPG (using a Schild analysis) in blocking the suppressant action of PPG was 5.95 (apparent K_b = 1.2 µM). The slope of the regression line from the Schild plot was 0.95 ± 0.19, which was significantly nonzero (p < 0.001). The correlation coefficient was r² = 0.683. These same concentrations of CPPG, however, were substantially less potent in blocking the suppressant action of MAP4 on 5-HT-induced EPSC (Fig. 9). In addition, CPPG alone produced a small, but statistically significant, increase in the frequency of 5-HT-induced EPSCs at the 30 µM concentration (p < 0.05), whereas the 100 µM concentration increased both the frequency and amplitude of 5-HT-induced EPSCs (p < 0.001; Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 7. CPPG blocks the suppressant action of PPG on 5-HT-induced EPSC. Top, the group III mGlu receptor antagonist CPPG blocks the suppressant action of PPG on 5-HT-induced EPSC in an apparent competitive manner (PPG alone, n = 5; PPG/CPPG 10 µM, n = 4; PPG/CPPG 30 µM, n = 3; PPG/CPPG 100 µM, n = 3). The 5-HT-induced EPSC frequency before PPG perfusion was 35.2 ± 6.2 N/s (basal); 24.1 ± 6.1 (10 µM CPPG); 22.4 ± 3.7 (30 µM CPPG); and 23.9 ± 1.8 (100 µM CPPG). Bottom, Schild regression analysis of the mGlu receptor antagonist CPPG for the PPG-induced suppression of 5-HT-induced EPSC. DR refers to the dose ratio and is the EC50 in the presence of the antagonist divided by the EC50 in the absence of the antagonist for the population of cells treated with PPG alone (n = 6). Linear regression yielded r = 0.686 and a slope of 0.946 ± 0.186. The pA2 value was –5.94. The Kb value calculated by Schild regression was 1.2 µM.

View larger version (31K): [in this window] [in a new window]   Fig. 9. MAP4 blocks suppressant action of L-AP4 on 5-HT-induced EPSCs and with minimal, if any, blockade of the MAP4 suppressant action by the preferential mGlu8 receptor antagonist CPPG. Top, MAP4 (300 µM) significantly suppresses 5-HT-induced EPSCs and also significantly attenuates the L-AP4 (30 µM)-induced suppression of 5-HT (100 µM)-induced EPSCs (n = 4). 5-HT-induced 17.8 ± 2.8 EPSC/s for this experiment. **, p < 0.01 compared with the suppressant effect of L-AP4 alone. Bottom, CPPG (10–100 µM, n = 4–5) administration did not result in a consistent dose-dependent attenuation of the suppressant action of MAP4.

The specificity of CPPG for mGlu receptors was further explored by testing the potency of 300 µM CPPG to block the suppressant action of LY354740 on 5-HT-induced EPSCs (data not shown). The mGlu2/3 receptor agonist LY354740 suppressed 5-HT-induced EPSCs with an EC₅₀ of 137 nM (n = 5). The pA₂ calculated by the Schild equation for CPPG antagonism of LY354740 agonist action was 4.12 (apparent K_b = 75.9 µM). Consistent with expected pharmacology, the mGlu2/3 receptor antagonist EGLU (400 nM, n = 5) returned apA₂ value of 6.64 (apparent K_b = 0.229 µM) against the suppressant action of LY354740 on 5-HT-induced EPSCs by using the Schild equation (not shown). In contrast, the pA₂ value calculated by using EGLU (200 µM, n = 5) to antagonize the suppressant action of PPG on 5-HT-induced EPSCs was 4.497 (apparent K_b = 33.1 µM; data not shown).

We then chose to test the mGlu receptor antagonist LY341495 against the suppressant action of the group III mGlu receptor agonists on 5-HT-induced EPSCs to isolate the effects of stimulating group III mGlu receptors from simultaneously stimulating group II mGlu receptors with endogenous glutamate. LY341495, while being a potent mGlu2/3 receptor antagonist (K_i = 0.0036 and 0.021 µM at mGlu3 and mGlu2, respectively), also blocks group III mGlu receptors over a wide range of concentrations (IC₅₀ = 0.173, 0.99, and 22.0 µM at mGlu8, mGlu7a, and mGlu4a, respectively) (Kingston et al., 1998). Therefore, we tested the ability of PPG to suppress the frequency of 5-HT-induced EPSCs following blockade of mGlu2/3/8 autoreceptors (Marek et al., 2000) and group III mGlu receptors with varying concentrations of LY341495 (0.1, 1, 10, and 30 µM). The lowest concentration of LY341495 tested (0.1 µM) is 4-fold greater than the K_i of the drug at mGlu2 receptors (Kingston et al., 1998) and 2-fold greater than the apparent K_b of the drug for blocking the suppressant action of the mGlu2/3 agonist LY354740 on 5-HT-induced EPSCs (Marek et al., 2000). It is likely that 0.1 µM LY341495, under our conditions, would be near or less than the concentration required to occupy 50% of mGlu8 receptors. As such, this concentration of the competitive antagonist would only be expected to shift the concentration-response determination for a group III agonist by less than 2-fold, if the agonist activates mGlu8 receptors to suppress the frequency of 5-HT-induced EPSCs. Surprisingly, this low concentration of LY341495 (0.1 µM) appeared to block the suppressant action of PPG in an "apparent" noncompetitive manner: the maximal effect of PPG seemed to be reduced to 50% suppression of 5-HT-induced EPSCs from >90% suppression without LY341495 (Fig. 8). Increasing the concentration of LY341495 by two orders of magnitude to 10 µM did not appreciably alter the maximal effect of PPG. We have previously shown that the mGlu receptor antagonist LY341495 (0.1–1 µM; see Table 2) significantly increased the frequency and amplitude of 5-HT-induced EPSCs, presumably by blocking mGlu2 autoreceptors (Marek et al., 2000). This finding would suggest that application of exogenous group III mGlu receptor agonists might act in concert with activation of group II mGlu receptors by endogenous glutamate being released by 5-HT_2A agonists. Activation of group III mGlu receptors would not necessarily be able to surmount the "apparent noncompetitive antagonism" exerted by the mGlu receptor antagonist at these low concentrations. However, higher concentrations of LY341495 did block, in a concentration-dependent manner, the suppressant action of PPG on the frequency of 5-HT-induced EPSCs (Fig. 8). Plotting the percentage blockade by LY341495 of the suppressant action of PPG (5 µM) on the frequency of 5-HT-induced EPSCs (using 52% suppression found for PPG in the presence of 100 nM LY341495 as the maximal effect) resulted in an extrapolated IC₅₀ = 38 µM.

View larger version (27K): [in this window] [in a new window]   Fig. 8. The effects of LY341495 (100 nM to 30 µM) and L-SOP (10–300 µM) or PPG (0.5–50 µM on 5-HT-induced EPSC. Top, the preferential mGlu2/3/8 antagonist LY341495 blocks the suppressant action of PPG on 5-HT-induced EPSC in an apparent noncompetitive manner (PPG alone, n = 5; PPG/LY341495, 100 nM, n = 6; PPG/LY341495, 1000 nM, n = 5; PPG/LY341495, 10,000 nM, n = 3; PPG/LY341495, 30,000 nM, n = 5). The 5-HT-induced EPSC frequency before PPG perfusion was 35.2 ± 6.2 (basal); 24.1 ± 6.1 (100 nM LY341495); 50.1 ± 3.8 (1000 nM LY341495); 35.9 ± 2.4 (10,000 LY341495); and 35.8 ± 5.02 N/s (30,000 nM LY341495). Bottom, the preferential mGlu2/3/8 antagonist LY341495 blocks the suppressant action of L-SOP on 5-HT-induced EPSC in an apparent noncompetitive manner (L-SOP alone, n = 5; L-SOP/LY341495, 100 nM, n = 5; L-SOP/LY341495, 1 µM, n = 5; L-SOP/LY341495, 10 µM, n = 3; L-SOP/LY341495, 30 µM, n = 4–6). The 5-HT-induced EPSC frequency before L-SOP perfusion was 31.7 ± 5.3 (basal); 37.0 ± 10.7 (100 nM LY341495); 35.1 ± 4.7 (1 µM LY341495); 26.1 ± 9.1 (10 µM LY341495); and 43.9 ± 10.3 N/s (30 µM LY341495).

A low concentration of LY341495 (0.1 µM) also seemed to block the suppressant action of L-SOP in an "apparent" noncompetitive manner: the maximal effect of L-SOP seemed to be reduced to 30 to 35% suppression of 5-HT-induced EPSCs (Fig. 8). Increasing the concentration of LY341495 by an order of magnitude to 1 µM did not appreciably alter the maximal effect of L-SOP. However, higher concentrations of LY341495 did block, in an apparent concentration-dependent manner, the suppressant action of L-SOP on the frequency of 5-HT-induced EPSCs (Fig. 8). Plotting the percentage blockade by LY341495 of the suppressant action of L-SOP (300 µM) on the frequency of 5-HT-induced EPSCs (using 30% suppression found for L-SOP in the presence of 100 nM LY341495 as the maximal effect) resulted in an extrapolated IC₅₀ = 42 µM. However, even 30 µM LY341495 blocked the suppressant action of 300 µM L-SOP on the 5-HT-induced EPSC frequency by only 31% (n = 5).

Because MAP4 has been suggested to be a partial agonist at mGlu4 receptors, the ability of MAP4 to block the suppressant effects of a group III mGlu receptor agonist was assessed. MAP4 (300 µM) exerted a significantly smaller suppressant effect on 5-HT-induced EPSCs than 30 µM L-AP4 (Fig. 9). Furthermore, whereas MAP4 (300 µM) suppressed 5-HT-induced EPSCs by 33.7%, MAP4 also significantly attenuated the suppressant action of L-AP4 (30 µM) on 5-HT-induced EPSCs (p < 0.05). As mentioned previously, CPPG did not potently block the suppressant action of MAP4 (Fig. 9), as was previously observed for the suppressant action of PPG (Fig. 7).

Because the apparent maximal efficacy of two group III mGlu receptor agonists in surmounting the effects of relatively low concentrations of LY341495 was attenuated, an additional mGlu2/3 antagonist (EGLU) was used to confirm that LY354740 could fully surmount the effects of an antagonist for the mGlu2 receptor. As described previously above, LY354740 suppressed 5-HT-induced EPSCs with an EC₅₀ = 137 nM (n = 5; not shown). The pA₂ value obtained from an experiment with 400 nM EGLU was 3.64 (apparent K_b = 229 µM). The maximal suppression of 5-HT-induced EPSCs by LY354740 was 88.9 ± 5.6 (1 µM LY354740) and 84.2 ± 4.4 (10 µM LY354740) in the absence and presence, respectively, of 400 µM EGLU. Thus, these results with EGLU and LY354740 were similar to those previously obtained with LY341495 and LY354740 (Marek et al., 2000).

Presynaptic Versus Postsynaptic Site of Action for Group III mGlu Receptor Agonists. The 5-HT-induced EPSCs are completely blocked by a selective AMPA antagonist (Zhang and Marek, 2000). If the effects of L-SOP were mediated by a postsynaptic action, then L-SOP might also be expected to block AMPA-induced steady-state inward currents in layer V pyramidal cells after treatment of the slice with TTX to block synaptic transmission. In these experiments, L-SOP (300 µM) did induce small outward currents (60 ± 20 pA, n = 5). However, L-SOP did not block the AMPA-induced inward currents (AMPA, 273 ± 58 pA; AMPA/L-SOP, 282 ± 67, n = 5; data not shown). In two of these five cells, the effects of L-SOP and AMPA following TTX treatment were similar when the drugs were prepared and applied to the slice in a nominally Ca²⁺ free artificial cerebrospinal fluid (data not shown).

The "apparent noncompetitive" blockade of the suppressant action of the group III mGlu receptor agonists by LY341495 may be secondary to the increased EPSC frequency and amplitude induced by 5-HT following blockade of presynaptic autoreceptors. On the other hand, such an action may simply be caused by relatively weaker efficacy of group III mGlu receptor activation at suppressing the increased frequency and amplitude of 5-HT-induced EPSCs that occurs following blockade of mGlu2/3 receptors. To examine this second possibility, we tested a drug that slows AMPA receptor desensitization. Cyclothiazide (50 µM x 20 min) slightly, but significantly, increased the frequency of 5-HT-induced EPSCs by 18.7% [t(4) = 3.48, p < 0.05; Fig. 10]. However, testing of individual cells found that cyclothiazide increased the EPSC frequency in only one of five cells (K-S test, p < 0.01). Cyclothiazide also increased the amplitude of 5-HT-induced EPSCs by 18.4% [t(4) = 3.66, p < 0.05; Fig. 10]. Testing of individual cells corroborated the significance of this finding as cyclothiazide increased the amplitude of 5-HT-induced EPSCs in four of five cells (K-S test, p < 0.01). Unlike in the LY341495 experiments and similar to the effects of L-SOP in the absence of an antagonist, L-SOP (300 µM) still suppressed the frequency of 5-HT-induced EPSCs by 66.6% following cyclothiazide (Fig. 10; compare with 65.9% suppression by L-SOP alone in Fig. 2). Thus, the decreased suppressant action of the group III mGlu receptor agonists following low concentrations of the mGlu2/3 receptor antagonist LY341495 does not seem to be simply caused by the inability of the group III mGlu receptor agonist in suppressing larger amplitude EPSCs.

View larger version (22K): [in this window] [in a new window]   Fig. 10. L-SOP suppresses 5-HT-induced EPSCs despite cyclothiazideinduced increase in EPSC frequency. Top, cyclothiazide (50 µM for 20 min) significantly increased the frequency of 5-HT-induced EPSCs by 18.4% (n = 5). L-SOP (300 µM) decreased 5-HT-induced EPSCs by 66.6% despite pretreatment with cyclothiazide. Cyclothiazide (bottom) also increased the amplitude of 5-HT-induced EPSCs. L-SOP decreased the amplitude of 5-HT-induced EPSCs following and during cyclothiazide treatment. *, p < 0.05 for respective comparison.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The main finding from the present studies is that group III mGlu receptor agonists preferentially suppress the frequency of 5-HT-induced EPSCs recorded from layer V pyramidal cells of the rat mPFC compared with electrically evoked EPSP or AMPA-induced EPSCs. Previously, we have shown that 5-HT induced an increase in EPSC frequency by selective activation of 5-HT_2A receptors (Aghajanian and Marek, 1997; Marek and Aghajanian, 1999). Recent studies involving medial thalamic chemical lesions and characterization of the suppressant effect of LY354740 on 5-HT-induced EPSCs have suggested that the midline and intralaminar thalamic nuclei are the site of origin for the majority of these afferents modulated by 5-HT_2A receptors (Marek et al., 2000). The present findings suggest that group III mGlu receptor agonists suppress 5-HT-induced EPSCs originating from the thalamocortical afferents.

Surprisingly, the pharmacological studies are consistent with the working hypothesis that activation of both mGlu8 and mGlu4 receptors suppress the 5-HT-induced EPSCs. First, the rank order of group III mGlu receptor agonists in suppressing the 5-HT-induced EPSCs agrees with their rank order in potency at mGlu4/mGlu8 receptors (De Colle et al., 2000). Second, the group III mGlu receptor agonist MAP4 suppressed the 5-HT-induced EPSCs with a potency suggesting mediation by mGlu4 receptors (De Colle et al., 2000). In contrast, the rank order potency of group III mGlu receptor agonists at suppressing 5-HT-induced EPSCs does not agree with the rank order potency of these drugs at mGlu6/mGlu7 receptors (Schoepp et al., 1999; Wright et al., 2000). Third, MAP4 is a partial agonist at the human mGlu4 receptor (Knopfel et al., 1995). The blockade by MAP4 of the suppressant action of L-AP4 against the 5-HT-induced EPSCs is consistent with this suggestion. Fourth, the high potency of PPG in suppressing 5-HT-induced EPSCs is not compatible with this drug activating only mGlu4 receptors, especially considering the Hill coefficient <1 and that PPG has been reported to be a mGlu8 receptor agonist with an 20-fold selectivity compared with mGlu4 receptors (Gasparini et al., 1999). Fifth, the mGlu8 receptor antagonist CPPG was >30-fold selective in blocking the suppressant action of PPG compared with MAP4, consistent with selectivity ratios for mGlu8 versus mGlu4 receptors (Naples and Hampson, 2001). Our estimate of the apparent K_b for CPPG against PPG (1.2 µM) agrees well with previous work (Thomas et al., 2001). Sixth, the potency of LY341495 in blocking the suppressant action of PPG and L-SOP (IC₅₀ > 30 µM) seems much closer to the affinity of LY341495 for mGlu4 receptors (IC₅₀ = 22 µM) than for mGlu8/mGlu7 receptors (IC₅₀ < 1 µM) (Kingston et al., 1998). Seventh, the potency of CPPG in blocking the suppressant action of MAP4 is similar to the potency of CPPG in blocking binding of L-AP4 to mGlu4 receptors (Han and Hampson, 1999). Eighth, mGlu4 receptor mRNA is especially strong in the intralaminar/midline thalamic nuclei (Ohishi et al., 1995; Neto et al., 2000) unlike the cortex (Ohishi et al., 1995; Neto et al., 2000). Ninth, mGlu4 receptor protein is enriched at the interface between layers IV and V of the neocortex (Phillips et al., 1997; Corti et al., 2002), which seems similar to the increased density of 5-HT_2A receptors in layer Va (Blue et al., 1988). Layer Va is also a major site of termination for thalamocortical afferents from the midline/intralaminar thalamic nuclei (Berendse and Groenewegen, 1991).

The second major finding was the selective suppression of 5-HT-induced EPSCs compared with the effects of endogenous excitatory amino acids on layer V pyramidal cells. L-SOP did not suppress electrically evoked EPSPs. Both L-AP4 and PPG exerted modest suppression of electrically evoked EPSPs using stimulation sites in the white matter underlying the cortex in contrast to robust suppression by group II mGlu receptor agonists (Marek et al., 2000). Others have previously observed 35 to 40% suppression by L-AP4 of electrically evoked EPSPs when recording from layer II/III, V, and VI pyramidal cells (Jin and Daw, 1998).

A significant source of the electrically evoked EPSPs from stimulation of the forceps minor probably is caused by antidromic activation of corticofugal projecting pyramidal cells from layers V and VI, such as corticostriatal pathways. Some controversy exists whether group III mGlu receptor agonists suppress EPSCs in striatal neurons as a result of activation of corticostriatal afferents (Lovinger and McCool, 1995; Pisani et al., 1997). It is noteworthy that mGlu7 receptors may be the presynaptic autoreceptor on corticostriatal afferents (Kosinski et al., 1999).

Another source of corticofugal projections is the pathway from layer VI pyramidal cells to primary thalamic relay nuclei. Again, the pharmacology with group III mGlu receptor agonists does not match the present results (Turner and Salt, 1999). Activation of mGlu7/8 receptors has been suggested to mediate suppression of corticothalamic EPSPs. The present electrical stimulation was performed in a "blind" fashion, and one cannot rule out the possibility that the proportion of corticostriatal versus corticothalamic axons might have differed between experiments with the relatively small number of cells tested.

Another striking observation was that a near maximal concentration of L-SOP (300 µM), which suppressed 5-HT-induced EPSCs, did not appreciably suppress AMPA-induced synaptic currents or the AMPA-induced steady-state inward current. Bath application of AMPA was used in the present experiments to release excitatory amino acids from synapses that terminate on the layer V pyramidal cells either by driving pyramidal cells to fire action potentials or activation of presynaptic AMPA receptors. Unlike the selective group III mGlu receptor agonists, the group II mGlu receptor agonist LY354740 did suppress the AMPA-induced EPSCs. In addition, L-SOP, unlike LY354740, was without effect on the AMPA-induced steady-state inward current. Differential efficacy for group III versus group II mGlu agonists in blocking AMPA-induced effects when AMPA receptors are desensitized by prolonged exposure to the slice represents one explanation for these observations. However, this does not explain the differential effects of group III versus mGlu II receptor agonists at electrically evoked EPSPs, which are primarily mediated by phasic AMPA receptor activation. Overall, these results are consistent with previous suggestions that the proconvulsant actions of group III mGlu agonists indicate a more prominent role for these mGlu receptors as heteroceptors on GABAergic terminals (Ghauri et al., 1996).

The group III mGlu receptor agonists probably suppressed 5-HT-induced EPSCs via a presynaptic action. First, concentrations of group III mGlu receptor agonists, which suppressed the frequency of 5-HT-induced EPSCs by 50%, generally did not decrease EPSC amplitudes. Changes in EPSC frequency suggest presynaptic effects, whereas changes in EPSC amplitude may result from either presynaptic or postsynaptic actions. Second, group III mGlu receptor agonists did not suppress the frequency or amplitude of basal EPSCs. Third, L-SOP was unable to block AMPA-induced EPSCs or steady-state inward currents (before and after TTX treatment). Fourth, group III mGlu receptor agonists showed minimal or no efficacy in suppressing electrically evoked EPSPs in layer V pyramidal cells. Fifth, the suppressant action of group III mGlu receptor agonists was attenuated by blockade of mGlu2/3 autoreceptors. Conversely, the suppressant action of a group III mGlu receptor agonist was not altered by a drug expected to have a postsynaptic action to slow AMPA receptor desensitization, cyclothiazide. Sixth, the group III mGlu receptor antagonist CPPG increased the frequency and amplitude of 5-HT-induced EPSCs consistent with an autoreceptor function. Taken together, these findings do not suggest a prominent role for postsynaptic group III mGlu receptors in mediating the suppression of 5-HT-induced EPSCs. However, a role for postsynaptic group III mGlu receptors on layer V pyramidal cells that are relatively restricted to dendritic compartments activated by glutamate released by 5-HT_2A receptor stimulation cannot be ruled out.

The "apparent" blockade of the maximal suppressant action of group III mGlu receptor agonists by the mGlu receptor antagonist LY341495, with particularly strong potency at mGlu2/3 receptors, is in stark contrast to our previous evidence that this mGlu receptor antagonist results in a rightward shift in the concentration-response curve for the mGlu2/3 receptor agonist LY354740 as expected for a competitive antagonist (Marek et al., 2000). Likewise, the mGlu2/3 receptor antagonist EGLU seemed to cause a rightward shift in the LY354740 concentration-response relationship consistent with competitive antagonism. Furthermore, the group III mGlu receptor antagonist CPPG seemed to exert a rightward shift in the concentration-response determination for the mGlu8 receptor agonist PPG consistent with competitive antagonism.

These findings of differential modes of antagonism for LY341495 against group III versus group II mGlu receptor agonists compared with the apparent competitive antagonism exerted by the group III mGlu receptor antagonist CPPG against group II or III mGlu receptor agonists, respectively, have two main implications. First, L-SOP and PPG seem to possess decreased efficacy in suppressing 5-HT-induced EPSCs following concentrations of the group II mGlu receptor antagonist, which increase 5-HT-induced EPSC frequency and amplitude. However, this effect can also be seen for a concentration of LY341495 (100 nM), which has minimal effects on the frequency and amplitude of 5-HT-induced EPSCs. These results suggest that the coupling of group III mGlu receptors to the suppression of 5-HT-induced EPSCs is weaker than that for group II mGlu receptors. Alternately, group III mGlu receptors may not be on an additional source of glutamatergic afferents for which 5-HT-induces EPSCs following thalamic lesions (Lambe and Aghajanian, 2001; Marek et al., 2001).

In summary, both group III and group II mGlu receptors seem to suppress 5-HT-induced EPSCs via a presynaptic mechanism. The presence of mGlu2, mGlu4, and/or mGlu8 receptor mRNA raises the possibility that the physiological interaction described in the present experiments provides a coincident detector function for the group III mGlu receptor in concert with mGlu2 receptors.

	Acknowledgements

 
We thank Drs. Eric Nisenbaum and Kurt Rasmussen for reviewing this manuscript.

	Footnotes

 
This work was supported by the United States Public Health Service Grants K08 MH01551 and R01 MH62186, a National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator Award, and the State of Connecticut (G.J.M.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.107490.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; mPFC, medial prefrontal cortex; mGlu2, metabotropic glutamate 2; EPSC, excitatory postsynaptic current; AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionate; LY293558, (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroquinoline-3-carboxylic acid; EPSP, excitatory postsynaptic potential; LY 300164, (R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4,5-h)(2,3)benzodiazepine; LY341495, 2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3(xanthy-9-yl)propanoic acid; L-AP4, L-4-phosphono-2-aminobutyric acid; L-SOP, L-serine-O-phosphate; PPG, (R,S)-4-phosphonophenylglycine; CPPG, (R,S)--cyclopropyl-4-phosphonophenylglycine; MAP4, (S)-2-amino-2-methyl-4-phosphonobutanoic acid; EGLU, (S)--ethylglutamic acid; TTX, tetrodotoxin; LY354740, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate; (1S,3S)ACPD, 1S,3S-1-aminocyclopentane-1,3-dicarboxylic acid.

Address correspondence to: Gerard J. Marek, Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Mail Drop 0510, Indianapolis, IN 46285. E-mail: gerard_j_marek{at}lilly.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Aghajanian GK and Marek GJ (1999) Serotonin-glutamate interactions: a new target for antipsychotic drugs. Neuropsychopharmacology 21: S122–S133.[CrossRef]

Aghajanian GK and Marek GJ (1997) Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Neuropharmacology 36: 589–599.[CrossRef][Medline]

Berendse HW and Groenewegen HJ (1991) Restricted cortical termination fields of the midline and intralaminar thalamic nuclei in the rat. Neuroscience 42: 73–102.[CrossRef][Medline]

Blue ME, Yagaloff KA, Mamounas LA, Hartig PR, and Molliver ME (1988) Correspondence between 5-HT₂ receptors and serotonergic axons in rat neocortex. Brain Res 453: 315–328.[CrossRef][Medline]

Corti C, Aldegheri L, Somogyi P, and Ferraguti F (2002) Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS. Neuroscience 110: 403–420.[CrossRef][Medline]

De Colle C, Bessis A-S, Bockaert J, Archer F, and Pin J-P (2000) Pharmacological characterization of the rat metabotropic glutamate receptor type 8a revealed strong similarities and slight differences with the type 4a receptor. Eur J Pharmacol 394: 17–26.[CrossRef][Medline]

Gasparini F, Bruno V, Battaglia G, Lukic S, Leonhardt T, Inderbitzin W, Laurie D, Sommer B, Varney MA, Hess SD, et al. (1999) (R,S)-4-phosphonophenylglycine, a potent and selective group III metabotropic glutamate receptor agonist, is anti-convulsive and neuroprotective in vivo. J Pharmacol Exp Ther 289: 1678–1687.[Abstract/Free Full Text]

Ghauri M, Chapman AG, and Meldrum BS (1996) Convulsant and anticonvulsant actions of agonists and antagonists of group III mGluRs. Neuroreport 7: 1469–1474.[Medline]

Han G and Hampson DR (1999) Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic receptor. J Biol Chem 274: 10008–10013.[Abstract/Free Full Text]

Jin X and Daw NW (1998) The group III metabotropic glutamate receptor agonist L-AP4, reduces EPSPs in some layers of rat visual cortex. Brain Res 797: 218–224.[CrossRef][Medline]

Kingston AE, Ornstein PL, Wright RA, Johnson BG, Mayne NG, Burnett JP, Belagaje R, Wu S, and Schoepp DD (1998) LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37: 1–12.[CrossRef][Medline]

Knopfel T, Lukic S, Leonardt T, Flor PJ, Kuhn R, and Gasparini F (1995) Pharmacological characterization of MCCG and MAP4 at the mGluR1b, mGluR2 and mGluR4a human metabotropic glutamate receptor subtypes. Neuropharmacology 34: 1099–1102.[CrossRef][Medline]

Kosinski CM, Bradley SR, Conn PJ, Levey AI, Landwehrmeyer GB, Penney JB Jr, Young AB, and Standaert DG (1999) Localization of metabotropic glutamate receptor 7 mRNA and mGlu7a protein in the rat basal ganglia. J Comp Neurol 415: 266–284.[CrossRef][Medline]

Lambe EK and Aghajanian GK (2001) The role of Kv1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex. J Neurosci 21: 9955–9963.[Abstract/Free Full Text]

Lambe EK and Aghajanian GK (2003) Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice. Neuron 40: 139–150.[CrossRef][Medline]

Lovinger DM and McCool BA (1995) Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGluR2 or 3. J Neurophysiol 73: 1076–1083.[Abstract/Free Full Text]

Mansour A, Fox CA, Burke S, Meng F, Thompson RC, Akil H, and Watson SJ (1994) Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J Comp Neurol 350: 412–438.[CrossRef][Medline]

Marek GJ and Aghajanian GK (1998) 5-HT-induced EPSCs in neocortical layer V pyramidal cells: suppression by µ-opiate receptor activation. Neuroscience 86: 485–497.[CrossRef][Medline]

Marek GJ and Aghajanian GK (1999) 5-HT_2A or ₁-adrenoceptor activation induces excitatory postsynaptic currents in layer V pyramidal cells of the medial prefrontal cortex. Eur J Pharmacol 367: 197–206.[CrossRef][Medline]

Marek GJ, Carpenter LL, McDougle CJ, and Price LH (2003) Synergistic action of 5-HT_2A antagonists and selective serotonin reuptake inhibitors in neuropsychiatric disorders. Neuropsychopharmacology 28: 402–412.[CrossRef][Medline]

Marek GJ, Wright RA, Gewirtz JC, and Schoepp DD (2001) A major role for thalamocortical afferents in serotonergic hallucinogen receptor function in neocortex. Neuroscience 105: 113–126.

Marek GJ, Wright RA, Schoepp DD, Monn JA, and Aghajanian GK (2000) Physiological antagonism between 5-hydroxytryptamine_2A and group II metabotropic glutamate receptors in prefrontal cortex. J Pharmacol Exp Ther 292: 76–87.[Abstract/Free Full Text]

McCormick DA, Connors BW, Lighthall JW, and Prince DA (1985) Comparative electrophysiology of pyramidal and sparsely spiny neurons of the neocortex. J Neurophysiol 54: 782–806.[Abstract/Free Full Text]

Moghaddam B and Adams BW (1998) Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonists in rats. Science (Wash DC) 281: 1349–1352.[Abstract/Free Full Text]

Muschamp JW, Regina MJ, Hull EM, Winter JC, and Rabin RA (2004) Lysergic acid diethylamide and (–)-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex. Brain Res 1023: 134–140.[CrossRef][Medline]

Naples MA and Hampson DR (2001) Pharmacological profiles of the metabotropic glutamate receptor ligands [³H]L-AP4 and [³H]CPPG. Neuropharmacology 40: 170–177.[CrossRef][Medline]

Neto FL, Schadrack J, Berthele A, Zieglgansberger W, Tolle TR, and Castro-Lopes JM (2000) Differential distribution of metabotropic glutamate receptor subtype mRNAs in the thalamus of the rat. Brain Res 854: 93–105.[CrossRef][Medline]

Ohishi H, Akazawa C, Shigemoto R, Nakanishi S, and Mizuno N (1995) Distributions of the mRNAs for L-2-amino-4-phosphonobutyrate-sensitive metabotropic glutamate receptors, mGluR4 and mGluR7, in the rat brain. J Comp Neurol 360: 555–570.[CrossRef][Medline]

Ohishi H, Shigemoto R, Nakanishi S, and Mizuno N (1993) Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53: 1009–1018.[CrossRef][Medline]

Phillips T, Makoff A, Brown S, Rees S, and Emson P (1997) Localization of mGluR4 protein in the rat cerebral cortex and hippocampus. Neuroreport 8: 3349–3354.[Medline]

Pisani A, Calabresi P, Centonze D, and Bernardi G (1997) Activation of group III metabotropic glutamate receptors depresses glutamatergic transmission at corticostriatal synapses. Neuropharmacology 36: 845–851.[CrossRef][Medline]

Saugstad JA, Kinzie JM, Shinohara MM, Segerson TP, and Westbrook GL (1997) Cloning and expression of rat metabotropic glutamate receptor 8 reveals a distinct pharmacological profile. J Pharmacol Exp Ther 51: 119–125.

Schoepp DD, Jane DE, and Monn JA (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431–1476.[CrossRef][Medline]

Scruggs JL, Signoracci TA, and Deutch AY (2000) DOI-induced activation of the cortex: dependence on 5-HT_2A heteroceptors on thalamocortical glutamatergic neurons. J Neurosci 20: 8846–8852.