5 s (25 bursts with 4 pulses per burst, for a total of 100 pulses. A DC holding current was injected to maintain the membrane potential at -70 mV, except during HFS or TBS when the potential was adjusted to -60 mV to facilitate spike firing. To calculate the paired-pulse ratio (PPR) two EPSCs were evoked with an inter-stimulus-interval of 50 ms, PPR was calculated as (eEPSC1/eEPSC2), where eEPSC1 and eEPSC2 represent the amplitude of the first and the second eEPSC, respectively. PPR was measured before, immediately after, as well as 10 min and 30 min after each of the LTP induction protocols (TBS, 5xHFS and 2xHFS). Drug application The following drugs were obtained from (1) Sigma-Aldrich (St. Louis, MO, USA): picrotoxin, 3-[[(3, 4-Dichlorophenyl) methyl] amino] propyl]diethoxymethyl) phosphinic acid (CGP52432); R-(+)-7-Chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-ben- zazepine (SCH23390 hydrochloride); 29, 59-Dideoxy-39-ATP; 49,5,7-Trihydroxyisoflavone; 5,7-Dihydroxy-3-(4-hydroxyphenyl)- 4H-1-benzopyran-4-one (genistein); TrkB/Fc Chimera human; (R)-N4-Hydroxy-N1-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]- 2-isobutyl succinamide (gallardin); Diethyldithiocarbamic acid (DETC); 7, 8-dihydroxyflavone hydrate; and BDNF. (2) Tocris Bioscience (Ellisville, MO, USA):1-(2-Diphenylmethoxyethyl)-4- (3-phenyl-2-propenyl)-piperazine dihydrochloride (GBR12783 dihydrochloride), (6)-1-Phenyl-2, 3, 4, 5-tetrahydro-(1H)-3- benzazepine-7, 8-diol hydrobromide (SKF38393 hydrobromide); (9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1- oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:39,29,19-kl]pyrrolo[3,4-i][1,6] benzodiazocine-10-carboxylic acid, hexyl ester (KT5720); and 1,4- Diamino-2,3-dicyano-1,4-bis[2-a minophenylthio]butadiene (U0126). All drugs were made as concentrated stock solutions in dH2O, except picrotoxin, genistein, KT5720, U0126, gallardin and 7, 8-dihydroxyflavone, which were made as stocks in 100% DMSO. The final concentration of DMSO was no more than 0.1% on final application. Drugs were applied in the ACSF using a continuous gravity fed bath application unless specifically stated. Statistics All data are expressed as the mean 6 SEM. The amplitudes of the EPSPs were normalized to the average baseline EPSP amplitude. All statistical tests were conducted using Excel 2007 or Graphpad Prism 4.0. Unless specified, the P value indicated for comparisons of the amplitude of LTP are determined using student’s t test on data obtained at 30-min post HFS. A two-way ANOVA was performed to test any effect of treatment and time on the access resistance or the membrane input resistance. A P,0.05 was considered statistically significant for all cases. Dual-immunofluorescence experiments Immunofluorescence experiments were performed as previously described [66]. Fluorescent immunohistochemistry was performed using the following primary antibodies: rabbit monoclonal anti- MMP17 (1:100, ab51075, Abcam, Cambridge, MA), rabbit monoclonal anti-MMP16 (1:100, ab73877, Abcam, Cambridge, MA), mouse monoclonal anti-CaMKIIa (1:1000, 05-532, Milli- pore, Lake Placid, NY), and rabbit polyclonal anti-TrkB antibody (1:1000, sc-12, Santa Cruz Biotechnology, Santa Cruz, CA). Co- localization of MMP17 with CaMKIIa and TrkB with CaMKIIa was determined by dual-immunofluorescent labeling. Representa- Dopamine and BDNF Modulate LTP in the BLA tive BLA sections (from Bregma -2.30 mm to -3.14 mm) were rinsed 3x (10 min each) in PBS, permeabilized with 0.5% Triton- X 100 in PBS, and incubated for 48 hrs at 4uC with two appropriate primary antibodies diluted in 0.5% Triton-X/PBS solution. Sections were rinsed 3x (10 min each) in PBS and then incubated at room temperature for 2 hrs with specific Alexa-Fluor secondary antibodies (1:500, Molecular Probes, Invitrogen, and Carlsbad, CA, USA), Alexa-Fluor 488 goat anti-mouse IgG and Alexa-Fluor 568 goat anti-rabbit IgG. Following incubation with secondary antibodies, sections were rinsed 3x (10 min each) in PBS and 1x in 0.05 M phosphate buffer (PB), mounted on gelatin- coated glass slides and coverslipped using Vectashield fluorescence mounting medium (Vector Laboratories, Inc., Burlingame, CA). Confocal spinning disk laser microscopy was used to analyze dual- immunofluorescence and to obtain high-resolution photomicro- graphs using an Orca R2 cooled CCD camera (Hammamatsu, Bridgewater, NJ) mounted on a Leica DM5500B microscope (Leica Mircosystems, Bannockburn, IL) equipped with a CSU10B Spinning Disk (Yokagawa Electronic Corporation, Tokyo, Japan). Antibody specificity The antibody against MMP-17 was developed from a synthetic peptide corresponding to residues on human MMP-17. MMP-17 antibody specificity was confirmed with Western blots by detection of a single band of approximately 58 kDa. MMP-16 antibody was developed against the synthetic peptide mapping at the C-terminal of human MMP16. Specificity of the antibody was confirmed with Western blots by detection of the single band at 57 kDa. The TrkB antibody was raised against a peptide mapping within the C- terminal cytoplasmic domain of Trk B of mouse origin. Specificity of the antibody was confirmed by the manufacturer (Santa Cruz Biotechnology) by detecting single band at 145 kDa, and has been confirmed previously [67]. The CaMKIIa antibody was devel- oped against a synthetic peptide corresponding to the N-terminus of rat CaMKIIa and its specificity has been described previously [68]. Single Cell RT-PCR Following recording, cell cytoplasm was aspirated into the recording pipette and expelled into a microcentrifuge tube. RT- PCR reactions were performed for testing mRNA expression using cDNA transcribed from individual cells. The RT product was then amplified in two cycles, the first amplification step using V3 (dT)24 primer and cycling conditions of 95uC for 3 min, 50uC for 2 min and 72uC for 3 min. For the second amplification step using V1 (dT)24 primer the cycling conditions were the same as mentioned above with 20-cycle PCR amplification and a 6s extension per cycle. The amplified cDNA from each cell was screened for the expression of 18S rRNA as a positive control marker. The amplified cDNA was subjected to another amplification step using 2 ml of cDNA from each cell as a template and 100 nM of each of the primers for D1, D5, TrkB, MMP17, ERK1/2 and CamKIIa. PCR was performed using a 10 min hot start at 95uC followed by a 40 cycle program (94uC for 40 sec, 56uC for 40 sec and 72uC for 1 min). Author Contributions Conceived and designed the experiments: CL DGR. Performed the experiments: CL JD RH. Analyzed the data: JD RH DGR. Wrote the paper: CL DGR. PLoS ONE | www.plosone.org 14 October 2011 | Volume 6 | Issue 10 | e26065