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SI Methods

The procedures used for slice preparation were approved by the Wayne State University animal investigation committee. Recordings were obtained either from acute slices or from slices maintained in culture for several days. The initial preparation procedure did not significantly differ for these two experimental preparations. Briefly, male Sprague-Dawley rats or mice (WT or 5-HT_2AR knockout littermates) (1, 2) aged either postnatal day 8 (P8) to P12 (for culturing), P15-P19, or older than P35 (adult rats, 100-150 g) were anaesthetized with halothane and killed by decapitation. The brain was quickly removed and cooled in ice-cold Ringer's solution of the following composition (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO₄, 2.5 CaCl₂, 1 NaH₂PO₄, 26.2 NaHCO₃, and 11 glucose, bubbled to saturation with 95% O₂/5% CO₂. The anterior pole of the brain was then isolated and affixed to a stage with cyanoacrylate glue. Coronal slices (300 mm thick) were cut by using a vibratome (Lancer series 1000; Ted Pella, Irvine, CA). Acute slices were transferred to a holding chamber where they were allowed to recover for at least 1 h in Ringer's solution. For culturing, slices were transferred to inserts and cultured essentially as previously described (2, 3).

The cDNA coding for pEGFP-N1 (GFP) was obtained from Clontech (Palo Alto, CA). 5-HT_2AR cDNA, subcloned in pRC/CMV vector (Invitrogen, Carlsbad, CA), was a gift from Dr. B. L. Roth (University of North Carolina, Chapel Hill, NC), whereas the dominant-negative form of PLCb1 fused to GFP (PLCb-CT subcloned into pEGFP-C1; Clontech) was a gift from Dr. S. R. Ikeda (National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD). Neuronal transfection was achieved by using particle-mediated gene transfer methods using a Bio-Rad Helios gene gun, as previously described (2, 3). The procedure used was essentially as recommended by the manufacturer, except for the use of a modified "gene gun barrel"(4) which allowed for lower pressures and less gold particles per "bullet" (one-half to one-quarter of the manufacturer's recommendation). Cortical slices were transfected (50-100 psi at 3-5 mm distance) with the appropriate set of bullets within 2 h after cutting. For the PLCb-CT experiment, bullets were coated with the plasmid coding for the PLCB-CT fused to GFP. For the 5-HT_2AR rescue experiments, bullets were coated with 5-HT_2AR plasmids and eGFP. We have previously shown that in >90% of cases, neurons that are effectively transfected express both plamids (3).

For recordings, slices were transferred to a recording chamber and superfused with Ringer's solution (in mM: 119 NaCl, 2.5 KCl, 1.3 MgSO₄, 2.5 CaCl₂, 1 NaH₂PO₄, 26.2 NaHCO₃, and 11 glucose, bubbled to saturation with 95% O₂/5% CO₂). Pyramidal cells were targeted using differential interference contrast (DIC) imaging on a fixed-stage upright microscope (BX50WI; Olympus). All whole-cell recordings from acute slices were obtained from layer V pyramidal neurons of the prelimbic or anterior cingulate subdivisions of the medial prefrontal cortex. For cultured slices, transfected cells were identified by fluorescence microscopy and targeted for recording by using DIC. Targeted pyramidal neurons were also generally restricted to layer V of this same region, although some neurons were from layer III, which, in this region, is adjacent to layer V. A small number of recordings were also obtained from comparable cells in the middle layers of somatosensory cortex. Because no obvious differences were observed between cells in these two areas, results were pooled for analysis. All recordings were obtained 2-7 days after transfection.

Electrical signals were recorded by using an Axopatch 200, Axopatch 200B amplifier, Axoclamp 2A/B (Axon Instruments, Foster City, CA) or with an EPC 10 (Heka, Lambrecht/Pfalz, Germany) and recorded online with the use of a paper-chart recorder (Gould 3400) and also digitized and stored in a PC. The recording pipettes were pulled from borosilicate glass (outer diameter, 1.2 mm) with the use of a Flaming-Brown horizontal puller to give resistance ranging from 3 to 6 MW. Three different internal solutions were used in the present study. All recordings from acute slices were carried out with the following (in mM): 115 potassium gluconate, 20 KCl, 2 MgCl₂, 10 Na₂ phoshocreatinine, 4 ATP, 0.3 GTP, and 10 Hepes. Two internal solutions were used for recordings from cultured slices. The first was (in mM): 120 potassium gluconate, 4 KCl, 2 MgCl₂, 10 Na₂ phoshocreatinine, 4 ATP, 0.3 GTP, and 10 Hepes. The second was (in mM): 115 KMeSO₄, 5 KCl, 5 NaCl, 0.02 EGTA, 1 MgCl₂, 10 Na₂ phosphocreatine, 4 ATP, 0.3 GTP, and 10 Hepes. The pH was adjusted to 7.3-7.4. In most experiments, series resistance was corrected by ≈60-70%. In the experiments involving recordings of miniature excitatory postsynaptic currents (mEPSCs; see below), series resistance was not compensated. Experiments in which access resistance increased by more than ≈30% were discarded. Some experiments involved simultaneous recordings from close neighboring cells: the maximal distance between these cells was ≈300 mm. In some experiments, the recorded neurons were filled with biocytin through the recorded pipette and visualized using a Vectastain peroxidase ABC kit (Vector Laboratories, Burlingame, CA)

Synaptic events were filtered at 1 or 3 kHz and digitized at 10 kHz by using a 12-bit A/D converter (Digidata 1200; Axon Instruments) under the control of pClamp 7.0 or 8.0 (Axon Instruments). Neurons were held at -70 mV, and spontaneous activity was sampled for 1 s every 6 or 10 s for the duration of the entire experiment. Even though, in acute slices, the vast majority of inward-going synaptic events induced by activation of 5-HT_2AR are glutamatergic (5), we minimized a possible contamination by GABAergic events by carrying out all recordings in cultured slices, with intracellular solutions bringing the reversal potential of chloride ions to around -60 or -72 mV. In the experiments involving recordings of mEPSCs, synaptic activity was continuously recorded for periods up to 20 min. For these experiments, slices were bathed in TTX (1-3 mM) and (-)bicuculline (30 mM). Analysis of synaptic events was carried out by using Mini Analysis Program (version 5.2; Synaptosoft, Leonia, NJ). Parameters for the synaptic event detection algorithm were optimized for every experiment. In simultaneous paired recordings from close neighboring cells, the detection algorithm was set on the noisiest of the recording and applied to both cells. In some of the recordings with GTPgS, the synaptic detection algorithm was determined during the presence of the agonist, when the current signal tended to become noisier, and applied to both cells for the entire duration of the experiments. The effects of 5-HT_2AR activation on the frequency and amplitude of spontaneous and miniature excitatory postsynaptic currents were plotted as interevent interval histograms and as amplitude histograms and cumulative amplitude histograms, respectively. Statistical significance was assessed by using the Kolmogorov-Smirnov (KS) statistical method or Student's t test, as indicated (P < 0.05 was taken as indicating statistical significance). All drugs were dissolved in Ringer's and bath-applied at known concentration.

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2. Villalobos C, Béïque JC, Gingrich JA, Andrade R (2005) Eur J Neurosci 22:1120-1126.

3. Béïque JC, Andrade R (2003) J Physiol 546:859-867.

4. O'Brien JA, Holt M, Whiteside M, Lummis SC, Hastings MH (2001) J Neurosci Methods 112:57-64.

5. Béïque JC, Chapin-Penick EM, Mladenovic L, Andrade R (2004) J Physiol 556:739-754.