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Previous Article | Next Article
The Journal of Neuroscience, December 15, 2001, 21(24):9856-9866
Control of Serotonergic Function in Medial Prefrontal Cortex by Serotonin-2A Receptors through a Glutamate-Dependent Mechanism
Raúl Martín-Ruiz1, M. Victoria Puig1, Pau Celada1, David A. Shapiro2, Bryan L. Roth2, Guadalupe Mengod1, and Francesc Artigas1 1 Department of Neurochemistry, Institut d' Investigacions Biomèdiques de Barcelona (Consejo Superior de Investigaciones Científicas), Institut d'Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain, and 2 Departments of Biochemistry, Neurosciences and Psychiatry, Case Western Reserve University Medical School, Cleveland, Ohio 44106
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
We examined the in vivo effects of the hallucinogen 4-iodo-2,5-dimethoxyamphetamine (DOI). DOI suppressed the firing rate of 7 of 12 dorsal raphe (DR) serotonergic (5-HT) neurons and partially inhibited the rest (ED50 = 20 µg/kg, i.v.), an effect reversed by M100907 (5-HT2A antagonist) and picrotoxinin (GABAA antagonist). DOI (1 mg/kg, s.c.) reduced the 5-HT release in medial prefrontal cortex (mPFC) to 33 ± 8% of baseline, an effect also antagonized by M100907. However, the local application of DOI in the mPFC increased 5-HT release (164 ± 6% at 100 µM), an effect antagonized by tetrodotoxin, M100907, and BAY × 3702 (5-HT1A agonist) but not by SB 242084 (5-HT2C antagonist). The 5-HT increase was also reversed by NBQX (AMPA-KA antagonist) and 1S,3S-ACPD (mGluR 2/3 agonist) but not by MK-801 (NMDA antagonist). AMPA mimicked the 5-HT elevation produced by DOI. Likewise, the electrical-chemical stimulation of thalamocortical afferents and the local inhibition of glutamate uptake increased the 5-HT release through AMPA receptors. DOI application in mPFC increased the firing rate of a subgroup of 5-HT neurons (5 of 10), indicating an enhanced output of pyramidal neurons. Dual-label fluorescence confocal microscopic studies demonstrated colocalization of 5-HT1A and 5-HT2A receptors on individual cortical pyramidal neurons.
Thus, DOI reduces the activity of ascending 5-HT neurons through a DR-based action and enhances serotonergic and glutamatergic transmission in mPFC through 5-HT2A and AMPA receptors. Because pyramidal neurons coexpress 5-HT1A and 5-HT2A receptors, DOI disrupts the balance between excitatory and inhibitory inputs and leads to an increased activity that may mediate its hallucinogenic action.
Key words: 5-hydroxytryptamine; 5-HT1A receptors; 5-HT2A receptors; AMPA; DOI; dorsal raphe nucleus; GABA; glutamate; hallucinogens; medial prefrontal cortex; microdialysis; mGluR; NMDA; single-unit recordings; thalamus
INTRODUCTION
The prefrontal cortex plays a crucial role in higher brain functions (Fuster, 1997
). It receives a dense innervation from the brainstem aminergic nuclei, including the serotonergic dorsal and median raphe nuclei of the midbrain (Azmitia and Segal, 1978
5-HT exerts complex actions on pyramidal neurons of the medial prefrontal cortex (mPFC). Previous work showed that 5-HT hyperpolarizes and depolarizes layer V pyramidal neurons by acting on 5-HT1A and 5-HT2 receptors, respectively (Araneda and Andrade, 1991
The apparent presence of 5-HT1A and 5-HT2A receptors in layer V pyramidal neurons suggests that they could be involved in the control of subcortical structures. The mPFC is one of the few forebrain areas projecting densely to the serotonergic dorsal raphe nucleus (DR) (Aghajanian and Wang, 1977
MATERIALS AND METHODS
Animals. Male albino Wistar rats (Iffa Credo, Lyon, France) weighing 280-320 gm and kept in a controlled environment (12 hr light/dark cycle and 22 ± 2°C room temperature) with food and water provided ad libitum, were used in in vivo experiments. Animal care followed the European Union regulations (O.J. of E.C. L358/1 18/12/1986).
Drugs and reagents. 5-HT oxalate, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT),
-amino-3-hydroxy-5-methyli-4-soxazole-4-propionate [(S)-AMPA],bicuculline, cyclothiazide, 1-[2,5-dimethoxy-4-iodophenyl-2-amino-propane (DOI), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), (+)MK-801 (dizolcipine), NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline, picrotoxininin, 6-chloro-5-methyl-1-[6-(2-methylpy-ridin-3-yloxy) pyridin-3-yl carbamoyl] indoline (SB 242084), tetrodotoxin (TTX), and N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide · 3HCl (WAY-100635), were from Sigma/Research Biochemicals (Natick, MA). 1S,3S-aminecyclopentane dicarboxylic acid (1S,3S-ACPD) was from Tocris (Bristol, UK). BAY × 3702, {R-(-)-2-237 4-[(chroman-2-ylmethyl)-amino]-butyl 253-1,1-dioxo-benzo[d]isothiazolone · HCl}, citalopram · HBr, LY 379268 {(
)-2-oxa-4-amino-bicyclo[3.1.0]hexane-4,6-dicarboxylate]}, and M100907 [R-(+)-
Surgery and microdialysis procedures. An updated description of the microdialysis procedures used can be found in Adell and Artigas (1998)
Three microdialysis experiments were conducted in chloral hydrate-anesthetized animals. In one of them, we examined the effect of the electrical stimulation of the midline thalamus projecting to mPFC, to modulate the release of 5-HT in the latter area. Microdialysis probes were implanted as above. Stimulating bipolar electrodes were used. The isolating material was peeled off ~1 mm above the tip, the poles were separated 100-150 µm, and electrodes were stereotaxically placed and secured with dental cement, as for microdialysis probes. The tip of the electrodes was aimed at the top part of the centromedial thalamic nucleus (AP
The second experiment examined the effects of the application of bicuculline in the midline thalamus (to disinhibit afferent fibers to mPFC) on the 5-HT release in mPFC. These rats were implanted with two dialysis probes, in mPFC (as above) and in the thalamus (coordinates: AP
The concentration of 5-HT in dialysate samples was determined by HPLC, as described (Adell and Artigas, 1998
Single-unit recordings. We examined the effect of the systemic administration of DOI on the firing activity of identified 5-HT neurons in the dorsal raphe nucleus (DR). Single-unit extracellular recordings were performed as previously described (Sawyer et al., 1985
. Descents were performed along the midline. Recorded 5-HT neurons were found 5.0-5.8 mm below the brain surface and were identified according to previously described electrophysiological criteria (Wang and Aghajanian, 1977
Single-unit potentials were amplified with a Neurodata IR283 (Cygnus Technology Inc., Delaware Water Gap, PA), postamplified, and filtered with a Cibertec amplifier (Madrid, Spain) and computed on-line using a DAT 1401plus interface system Spike2 software (Cambridge Electronic Design, Cambridge, UK). After recording stable baseline spontaneous activity for at least 5 min, DOI was administered (0.025-0.4 mg/kg, i.v.; cumulative doses) every 2 min. Once a maximal effect of DOI was attained, either M100907 (100 µg/kg, i.v.; Haddjeri et al., 1999
Additional experiments were performed to examine the effects of the local application of DOI in mPFC on the firing rate of 5-HT neurons in the DR. DOI (200 µM, dissolved in aCSF) was infused through a 32 gauge stainless steel cannula (Small Parts Inc., Miami, FL) implanted in the mPFC (tip coordinates: AP +3.4, L
Data and statistical analysis. Microdialysis results are expressed as femtomoles per fraction (uncorrected for recovery) and shown in figures as percentages of basal values (individual means of four predrug fractions). Statistical analysis of drug effects on dialysate 5-HT was performed using one- or two-way ANOVA for repeated measures of raw data with time as repeated factor and dose or pretreatment as independent factor. The effects of drugs or chemical stimulation on the 5-HT output were analyzed by one- or two-way ANOVA of raw data followed by post hoc Duncan test. Changes in firing rate were quantified by averaging the values in the second minute after drug injection and expressed as percentage of baseline. ED50 values were calculated with the GraphPad (San Diego, CA) Prism program. Data are expressed as the mean ± SEM. Statistical significance has been set at the 95% confidence level (two-tailed).
Cell culture and monoclonal antibody production. Stably transfected Chinese Hamster Ovary (CHO) cells expressing the 5-HT1A receptor were obtained from J. Raymond (Medical University of South Carolina, Charleston, SC). A monoclonal 5-HT1A receptor antibody was prepared as follows. Female BALB/c mice were immunized intraperitoneally with 50 µg of a synthetic peptide (NKRTPRRAAALISLC) conjugated to Limulus polyphemus hemocyanin and emulsified in Freund's adjuvant. Immune spleens were harvested after ~3 months and fused with SP2/0 myeloma cells at a ratio of four spleen cells per myeloma using phenylethyleneglycol. Antibody-producing colonies were detected by ELISA as follows: immunizing peptide conjugated to BSA (10 µg/ml) was immobilized to COSTAR 9017 EIA/RIA plates in 100 µl of coating buffer (50 mM carbonate-bicarbonate, pH 9.6) and incubated overnight at room temperature. Wells were blocked with 2% milk in PBS (blocking buffer) 1 hr, followed by addition of antibody in culture supernatant or in blocking buffer for 90 min. Antibodies were detected using a horse anti-mouse polyclonal antibody linked to horseradish peroxidase (Vector Laboratories, Burlingame, CA; 1:2000 in blocking buffer) followed by spectrophotometric detection at 405 nm using the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate system (Sigma, St. Louis, MO). Hybridomas were subjected to three rounds of limiting dilution cloning, after which antibody was produced in bulk by generation of ascites in histocompatible mice. Immunoreactivity of the antibodies was confirmed both by ELISA (as described above) and by immunohistochemical staining using CHO cells (control) or CHO cells stably expressing the 5-HT1A receptor. Briefly, cells on microscope coverslips in 24 well microtiter plates were serum-starved overnight at 37°C and 5% CO2, then fixed in 4% paraformaldehyde and maintained at room temperature. Cells were permeabilized 20 min with 0.1% Triton X-100 and blocked with 2% milk for 1 hr. Anti-5-HT1A antibody supernatants were incubated with the cells for 90 min, followed by incubation for 1 hr with a Texas Red-labeled goat anti-mouse secondary reagent (Vector Laboratories). Cells were then mounted for confocal microscopy as previously described (Willins et al., 1997
Confocal immunofluorescence studies. Rats were prepared for immunofluorescence studies as previously detailed (Willins et al., 1997
Dual-label confocal microscopy was performed using a Zeiss Model 410 confocal microscope as previously described (Willins et al., 1997
RESULTS
Baseline 5-HT values
The basal concentration of 5-HT in dialysates from medial prefrontal cortex was 17.5 ± 0.8 fmol/fraction (n = 105) in freely moving rats and 17.0 ± 1.1 fmol/fraction (n = 24) in chloral hydrate-anesthetized rats.
Effects of the local application of DOI in medial prefrontal cortex
The local application of 1, 10, and 100 µM DOI (2 hr each) increased the 5-HT output in a concentration-dependent manner. Maximal increases at each concentration were, respectively 119 ± 20, 137 ± 16, and 209 ± 29% of baseline (n = 5) (Fig. 1A). One-way ANOVA showed a significant effect of DOI application (p < 0.00001). In subsequent experiments, we used the concentration of DOI that elicited maximal effects (100 µM).
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Figure 1. A, Concentration-dependent increase of 5-HT in dialysates from medial prefrontal cortex evoked by the local application of 1, 10, and 100 µM of the partial 5-HT2A/2C receptor agonist DOI (filled circles; n = 5). The bar graph in the inset shows the average increase in 5-HT obtained from the last four (stabilized) fractions at each DOI concentration (+p < 0.05 vs baseline). B, Effect of the continuous application of 100 µM DOI (filled squares; n = 7). Control rats (n = 6) received artificial CSF throughout the experiment. Bars show the period of drug application. +p < 0.05 versus baseline; Duncan test post-ANOVA.
The perfusion of 100 µM DOI (n = 7) significantly increased the 5-HT output when compared with the control group (artificial CSF; n = 7) (p < 0.02, treatment effect; p < 0.00001, time effect; p < 0.00001 time × treatment interaction) (Fig. 1B). Maximally elevated 5-HT values were observed soon (40 min) after DOI application and remained elevated over the entire period of application. The mean increase produced by 100 µM DOI in all perfusion experiments was 164 ± 6% of baseline (average of fractions 7-10; n = 69).
The effect of DOI was completely antagonized by the coperfusion of 1 µM TTX, which reduced 5-HT values below baseline (from 185 ± 10 to 30 ± 6% of baseline; n = 4; p < 0.00001) (Fig. 2). Likewise, the 5-HT increase produced by 100 µM DOI was significantly attenuated by the coperfusion of 100 µM of the selective 5-HT2A receptor antagonist M100907 (DOI: 181 ± 8% of baseline; DOI + M100907: 119 ± 13% of baseline; n = 8; p < 0.00001) (Fig. 3A). A higher M100907 concentration (300 µM) elicited a greater reduction of the 5-HT output (from 163 ± 1% to 42 ± 6% of baseline, n = 6; p < 0.00001) (Fig. 3A). In contrast, the selective 5-HT2C receptor antagonist SB 242084 did not reverse the 5-HT elevation produced by DOI and increased the 5-HT output further, from 178 ± 22% to 224 ± 25% of baseline (n = 6; p < 0.00001) (Fig. 3B).
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Figure 2. The perfusion of tetrodotoxin (TTX; 1 µM) reversed the increase in 5-HT output induced by the local application of DOI 100 µM (n = 4). Bars show the period of drug application. The effect of DOI alone is shown by a dotted line. +p < 0.05 versus baseline (DOI alone); *p < 0.05 versus DOI alone (DOI + drug); Duncan test post-ANOVA.
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Figure 3. A, The application of the selective 5-HT2A receptor antagonist M100907 (100 and 300 µM; filled triangles and circles, n = 8 and 4, respectively) antagonized the increase in 5-HT output induced by the local application of DOI 100 µM. B, Lack of antagonism of the effect of DOI by the selective 5-HT2C receptor antagonist SB 242084 (100 µM; filled circles, n = 6). This drug significantly potentiated the 5-HT increase elicited by DOI (shown by a dotted line). +p < 0.05 versus baseline (DOI alone); *p < 0.05 versus DOI alone (DOI + drug); Duncan test post-ANOVA.
The perfusion of the noncompetitive NMDA receptor antagonist MK-801 (300 µM) did not significantly modify the effect of DOI (n = 7) (Fig. 4A). In contrast, the application of the AMPA-KA receptor antagonist NBQX (300 µM) significantly reduced the effect of DOI (from 182 ± 15% to 122 ± 13% of baseline; n = 6; p < 0.00001) (Fig. 4B). The perfusion of the preferential mGluR 2/3 agonist 1S,3S-ACPD (300 µM) modestly reversed the effect of DOI on 5-HT output only in the last two fractions (n = 5; p < 0.00001) (Fig. 4C). A higher concentration of 1S,3S-ACPD (1 mM) elicited a more marked antagonism and almost completely reversed the effect of DOI, from 190 ± 15 to 112 ± 6% of baseline (n = 5; p < 0.00001) (Fig. 4C). In contrast, the application of the selective mGluR 2/3 agonist LY 379268, perfused at three different concentrations (300 µM, 1 mM, and 3 mM) failed to significantly reduce the 5-HT increase induced by the local application of DOI. The average effect of LY 379268 was 96 ± 16, 97 ± 12, and 107 ± 10% at 300 µM, 1 mM, and 3 mM, respectively (n = 4-5, data expressed as percentage of the 5-HT values during DOI alone). The effect of DOI was mimicked by the local application of AMPA (300 µM), which induced a maximal elevation of 5-HT to 188 ± 15% of baseline (p < 0.001, treatment effect; p < 0.00001, time effect; p < 0.00001, time × treatment interaction compared with controls; n = 6) (Fig. 4D).
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Figure 4. A, The application of the noncompetitive NMDA receptor antagonist MK-801 (300 µM) did not reverse the increase in 5-HT output induced by DOI 100 µM (n = 7). B, Antagonism by the AMPA-KA antagonist NBQX (300 µM) of the effect of DOI (n = 7). C, The mGluR 2/3 agonist 1S,3S-ACPD (300 µM, filled triangles; 1 mM, filled circles; n = 5 each) reversed the 5-HT elevation induced by DOI. D, The perfusion of 300 µM AMPA elevated the 5-HT similarly to DOI (n = 6; filled circles). The effect of DOI alone is shown by a dotted line. +p < 0.05 versus baseline (DOI or AMPA); *p < 0.05 versus DOI alone (DOI + drug); Duncan test post-ANOVA.
The increase in 5-HT output produced by the application of DOI was strongly antagonized by the coperfusion of the selective 5-HT1A receptor agonist BAY × 3702 (De Vry et al., 1998
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Figure 5. The selective 5-HT1A receptor agonist BAY × 3702 (30 µM) reversed the DOI-induced increase in 5-HT output (n = 5). The effect of DOI alone is shown by a dotted line. +p < 0.05 versus baseline (DOI alone); *p < 0.05 versus DOI alone (DOI + drug); Duncan test post-ANOVA.
Localization of 5-HT1A and 5-HT2A receptors on individual mPFC pyramidal neurons
The opposite effects of 5-HT1A and 5-HT2A receptor activation in mPFC on 5-HT release are in keeping with previous data on pyramidal neuron excitability (Araneda and Andrade, 1991
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Figure 6. Colocalization of 5-HT2A and 5-HT1A receptors in individual cortical neurons. A shows a representative mPFC section depicting individual cortical neurons expressing 5-HT1A-like immunoreactivity; B shows the same section with 5-HT2A-like immunoreactivity; C depicts an overlay showing colocalization of 5-HT2A and 5-HT1A receptor-like immunoreactivity in individual cortical neurons. Magnification, 400×. D shows a high-power (1000×) view of individual cortical neurons expressing 5-HT1A-like immunoreactivity; E shows the same section with 5-HT2A-like immunoreactivity; F depicts an overlay of 5-HT1A and 5-HT2Alike immunoreactivity. G shows a low-power (400×) view of cortical neurons visualized with a guinea pig 5-HT1A receptor antibody, whereas H shows the same section visualized with a monoclonal 5-HT2A receptor antibody, with colocalization indicated by arrowheads. I shows a typical section incubated only with secondary antibodies (400×).
Effects of increased glutamatergic transmission in mPFC on serotonergic function
The above microdialysis results suggested that DOI increased 5-HT release in mPFC indirectly, possibly by (1) enhancing a glutamatergic tone on 5-HT nerve terminals (local effect) and/or (2) by enhancing the activity of ascending 5-HT neurons in the DR through descending excitatory inputs. We therefore examined the effects of directly increasing the glutamatergic activity in mPFC on the local 5-HT release.
We first studied whether the 5-HT-increasing effects of DOI could be mimicked by endogenous glutamate. To this end, we electrically stimulated the mediodorsal and centromedial thalamic nuclei (5 Hz, 1.5 mA, 0.3 msec) for 20 min and collected dialysate fractions from the ipsilateral mPFC. The electrical stimulation of these nuclei elevated the 5-HT output in mPFC to a maximum of 179 ± 31% of baseline (p < 0.00001; n = 6) (Fig. 7A). Sham stimulation did not alter 5-HT levels (n = 6) (Fig. 7A). Likewise, in dual probe microdialysis experiments, the application of bicuculline (300 µM and 1 mM) by reverse dialysis in the mediodorsal and centromedial thalamic nuclei doubled the 5-HT release in mPFC (n = 8; p < 0.0007) (Fig. 7B).
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Figure 7. A, The electrical stimulation of a midline thalamic area affecting the mediodorsal nucleus and a small part of the centromedial nucleus (5 Hz, 1.5 mA, 0.3 msec pulses; shown by a bar) for 20 min increased the 5-HT release in the mPFC of chloral hydrate-anesthetized rats (filled triangles; n = 6). Control rats were implanted with microdialysis probes and thalamic electrodes, but no current was passed (n = 6; open circles). *p < 0.05 versus baseline; Duncan test post-ANOVA. B, In dual probe microdialysis experiments, the application of bicuculline (BIC; 300 µM and 1 mM, shown by a bar; n = 8) by reverse dialysis in the centromedial and mediodorsal thalamic nuclei significantly increased the 5-HT release in mPFC. The variability of the 5-HT increase was relatively large, possibly because of slight differences in the location of probes. *p < 0.05 versus baseline; Duncan test post-ANOVA. C, Effects of the local application of the glutamate uptake inhibitor L-trans-PDC, alone or in combination with NBQX, MK-801, and cyclothiazide on the 5-HT release in mPFC (n = 4-5). L-trans-PDC (4 mM) significantly increased the 5-HT release. This effect was prevented by the coperfusion of NBQX but not by MK-801 (300 µM each). Cyclothiazide (300 µM), a drug that prevents the desensitization of AMPA receptors, did not affect the time course or potentiate the effect of L-trans-PDC, suggesting that the marked tachiphylaxis of the effect is possibly caused by a presynaptic adaptive change in glutamate synthesis and release resulting from prolonged inhibition of reuptake. +p < 0.05 versus baseline; *p < 0.05 versus L-trans-PDC alone; Duncan test post-ANOVA.
Moreover, the local (in mPFC) application of the glutamate uptake inhibitor L-trans-PDC markedly increased the local 5-HT release (n = 4; p < 0.000001) (Fig. 7C). The time course of the effect differed from that elicited by AMPA (Fig. 4D) and showed a marked tachyphylaxis. However, the 5-HT increase was fully prevented by the coapplication of 300 µM NBQX (p < 0.00005 vs L-trans-PDC alone) (Fig. 7C) but not by 300 µM MK-801, thus supporting the involvement of AMPA-KA receptors. The coapplication of 300 µM cyclothiazide to prevent a putative desensitization of the glutamate receptors involved in this effect failed to modify the effect of L-trans-PDC, suggesting that the transient nature of the 5-HT increase was likely attributable to presynaptic adaptive mechanisms after glutamate uptake blockade.
Effects of DOI on the firing rate of 5-HT neurons in the DR
The systemic administration of DOI to chloral hydrate-anesthetized rats induced a dose-dependent reduction of the firing rate of 5-HT neurons in the DR (Fig. 8). Two putative subgroups of neurons were identified, according to the maximal effect of DOI. In 7 of 12 neurons recorded, DOI fully suppressed the serotonergic activity at low doses. In the rest (n = 5), the effect of DOI on serotonergic cell firing was partial, with a maximal decrease to ~40% of baseline. The administration of additional doses of DOI did not result in a further reduction. In some neurons, higher doses of DOI appeared to elicit a slight increase in firing rate, as shown in Figure 8B. In both subgroups, the effect of DOI was reversed by the administration of M100907 (100 µg/kg, i.v.) (Fig. 8B,D). The calculated ED50 value was 20 µg/kg intravenously for all neurons (19 and 22 µg/kg, i.v., for neurons with full and partial inhibitory response, respectively). When examined, the administration of the 5-HT1A receptor agonist 8-OH-DPAT suppressed serotonergic cell firing in both subgroups of neurons (Fig. 8C,D). The further administration of WAY 100635 (10 µg/kg, i.v.) returned firing rate to baseline (Fig. 8D). The inhibitory effect of DOI on serotonergic cell firing was also reversed by the administration of the GABAA receptor antagonist picrotoxinin (1 mg/kg, i.v.) (Fig. 8E).
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Figure 8. Effects of the intravenous administration of DOI on serotonergic neurons of the DR. A-E, Integrated firing rate histograms showing the effect of DOI on five different 5-HT neurons. A-C depict neurons with full (A) and partial (B, C) inhibitory responses to the administration of cumulative doses of DOI (25-50 µg/kg, i.v. in A; 25-400 µg/kg, i.v. in B, C). The effect of DOI was antagonized by M100907 (100 µg/kg, i.v.; examples in B and D). The serotonergic nature of neurons with partial or full response to DOI is illustrated by its sensitivity to 8-OH-DPAT (C, D). D shows the inhibitory effect of DOI (25-50 µg/kg, i.v.). After reversal by M100907 (100 µg/kg, i.v.), 8-OH-DPAT (DPAT; 0.25-2 µg/kg, i.v.) fully suppressed firing activity, and the 5-HT1A receptor antagonist WAY 100635 (WAY; 10 µg/kg, i.v.) returned firing rate to baseline. E shows the reversal of the effect of DOI (25-200 µg/kg, i.v.) by the GABAA antagonist picrotoxinin (PTX; 1 mg/kg, i.v.). The graph in F shows the dose-response curves for DOI in 5-HT neurons. The neurons with partial and full inhibitory responses to DOI administration had comparable ED50 values (19 and 22 µg/kg, i.v.; n = 7 and 5, respectively). The dotted line shows the dose-response curve for all neurons (n = 12).
To assess whether the increased mPFC release of 5-HT induced by local DOI application was caused by the activation of pyramidal neurons projecting to the DR (thus enhancing the activity of ascending 5-HT neurons), we locally infused DOI in the mPFC while recording 5-HT neurons in the DR. In 5 of 10 serotonergic neurons in the DR, the application of DOI in mPFC (200 µM, 200 nl applied in 1 min) enhanced their firing rate (to 194 ± 45% of baseline; range, 137-371%; p < 0.02) (Fig. 9A). One additional neuron had a marginal increase (115% of baseline), and the rest were unaffected by the application of DOI in mPFC (Fig. 9B). The effects of a second dose of DOI (5 min after the first one) were examined in seven neurons. Of these, three responded with a further increase in DR firing (from 155 to 260% of baseline). The infusion of vehicle in mPFC did not affect the firing rate of DR 5-HT neurons (data not shown).
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Figure 9. Integrated firing rate histograms of DR serotonergic neurons after the application of DOI in mPFC (200 µM, 200 nl, shown by a 1 min bar). The neuron in A responded with an increase in firing rate after the first and second application of DOI (note the persistence of the increased firing rate), whereas the neuron in B was unaffected by the application of two consecutive doses of DOI. The distal effect of DOI on 5-HT cell firing cannot be accounted for by drug diffusion to the DR because of the small amount of tissue affected by DOI application (0.2 µl) and the inhibitory effect of DOI on 5-HT neurons when acting on DR 5-HT2A receptors (Liu et al., 2000
Effect of the systemic administration of DOI on 5-HT release in medial prefrontal cortex
The systemic administration of DOI (1 mg/kg, s.c.) to chloral hydrate-anesthetized rats reduced the extracellular 5-HT concentration in the mPFC to a maximal effect of 33 ± 8% of baseline 2 hr after administration (n = 4; p < 0.00001) (Fig. 10). The effect of DOI was reversed by the administration of M100907 (0.5 mg/kg, s.c.) which returned 5-HT levels to above baseline (127 ± 6%; p < 0.00001) (Fig. 10).
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Figure 10. The systemic administration of DOI (1 mg/kg, s.c.) markedly reduced the 5-HT output in mPFC of chloral hydrate-anesthetized rats (n = 4). The selective 5-HT2A receptor antagonist M100907 (0.5 mg/kg, s.c.) fully reversed the effect of DOI. Arrows mark injections. +p < 0.05 versus baseline; *p < 0.05 versus DOI alone; Duncan test post-ANOVA.
DISCUSSION
The present results show that the stimulation of 5-HT2A receptors by the hallucinogen DOI markedly affects serotonergic transmission. Systemic DOI administration reduced the activity of DR 5-HT neurons and their release by mPFC terminals. Conversely, the selective activation of prefrontal 5-HT2A receptors raised the local 5-HT release and the firing of a subgroup of 5-HT neurons.
Despite the low density of 5-HT2A receptors in the DR and adjacent areas (Pompeiano et al., 1994
Unlike its systemic administration (Wright et al., 1990
Several lines of evidence suggest that DOI increases the 5-HT release in mPFC indirectly, through the stimulation of glutamate release and subsequent activation of AMPA-KA receptors (Fig. 11). First, the 5-HT increase was reversed by NBQX but not by MK-801 and was mimicked by AMPA application. Second, several strategies aimed at increasing glutamatergic neurotransmission in the mPFC, such as the perfusion of the glutamate reuptake inhibitor L-trans-PDC (also enhancing striatal 5-HT release; Abellán et al., 2000
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Figure 11. Scheme of the putative presynaptic and postsynaptic actions of DOI. In the mPFC, DOI increases 5-HT release. This effect involves the activation of prefrontal 5-HT2A receptors, possibly on glutamatergic afferents from midline thalamus. The role of 5-HT2A receptors on pyramidal neurons and GABA interneurons has not been elucidated. The activation of prefrontal 5-HT2A receptors enhances the release of glutamate, which acts on pyramidal AMPA receptors and increases impulse flow in layer V pyramidal neurons projecting to the DR, thus resulting in an increased serotonergic activity and 5-HT release in mPFC. Additionally, glutamate might activate terminal AMPA-KA receptors in 5-HT terminals and increase 5-HT release in a local manner. This might imply axo-axonic contacts between glutamatergic and serotonergic axons, not yet established anatomically but for which there is functional evidence (see Discussion). The activation of inhibitory 5-HT1A receptors on pyramidal neurons counteracts the effects of DOI and decreases 5-HT release, likely by reducing DOI excitatory effects on descending inputs to the DR, because the selective activation of prefrontal 5-HT1A receptors reduces the firing rate of DR 5-HT neurons (Celada et al., 2001
A critical step in the proposed mechanism is the actual assessment of the effect of DOI on glutamate release. However, extracellular glutamate concentrations are not representative of the transmitter-synaptic pool (Timmerman and Westerink, 1997
The present results accord with previous in vitro work in mPFC slices showing that the activation of local 5-HT2A receptors increases spontaneous EPSCs in layer V pyramidal neurons by an AMPA-dependent mechanism (Aghajanian and Marek, 1997
The cellular source or sources of glutamate and the precise localization or localizations of 5-HT2A receptors accounting for this effect are debated. 5-HT2A receptors occur in three different populations in frontal cortex: pyramidal neurons (major 5-HT2A-containing neurons), GABAergic interneurons, and axon terminals (Willins et al., 1997
Indeed, a preferential action of DOI on terminal 5-HT2A receptors would leave the dense population of pyramidal 5-HT2A receptors without a significant role. Interestingly, the DOI-induced increase in 5-HT release was potently counteracted by coperfusion of the selective 5-HT1A receptor agonist BAY × 3702. 5-HT1A and 5-HT2A receptors colocalize on cortical pyramidal neurons (Fig. 6A-I) and have opposite effects on their excitability (Araneda and Andrade, 1991
5-HT2A receptors play a crucial role in the filtering of inputs reaching the somata of pyramidal neurons because their activation increases neuronal excitability. The activation by DOI of prefrontal 5-HT2A receptors increases the excitability of pyramidal neurons. At the same time, its action on (possibly) midbrain 5-HT2A receptors (Liu et al., 2000
Further work is required to clarify the relative role of local and distal mechanisms in the control of 5-HT release by prefrontal 5-HT1A/2A receptors and the precise localization of 5-HT2A receptors responsible for this effect of DOI.
FOOTNOTES Received July 9, 2001; revised Sept. 18, 2001; accepted Sept. 26, 2001.
This work was supported by National Institutes of Health Grants FIS 01/1147 and SAF01-2133 (F.A.), KO2MH01366 and RO1MH61887 (B.L.R.), and a National Alliance for Research on Schizophrenia and Depression Young Investigator Award to D.A.S. M.V.P. is recipient of a predoctoral fellowship from the Institut d'Investigacions Biomèdiques August Pi i Sunyer. Financial support from Bayer S.A. is also acknowledged. We thank the pharmaceutical companies for the generous supply of drugs. The technical help of Leticia Campa is gratefully acknowledged.
R.M.R. and M.V.P. contributed equally to this work.
Correspondence should be addressed to Dr. Francesc Artigas, Department of Neurochemistry, Institut d' Investigacions Biomèdiques de Barcelona (Consejo Superior de Investigaciones Científicas), Institut d'Investigacions Biomèdiques August Pi i Sunyer, Rosselló, 161, Sixth floor, 08036 Barcelona, Spain. E-mail: fapnqi{at}iibb.csic.es.
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