A complete listing of reagents is provided in Supplemental Material, p. 3
). Animals were treated humanely and with regard for alleviation of suffering according to protocols approved by the Institutional Animal Care and Use Committees of Oregon Health & Science University, University of California, Davis, and Washington State University, Pullman.
Hippocampal neurons were dissociated from postnatal day–1 Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) and cultured at high density (105
) in Neurobasal-A medium (Invitrogen, Carlsbad, CA) supplemented with B27 (Invitrogen) as described previously (Wayman et al. 2006
). To visualize dendritic arbors, cultures were transfected at 6 days in vitro
(DIV) with the plasmid-encoding microtubule-associated-protein-2B MAP2B (which labels the somatodendritic domain) fused to enhanced green fluorescent protein (EGFP) using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s protocol. A subset of cultures was simultaneously transfected with plasmids encoding dominant negative (dn) CaMKI (dnCaMKI), dnCREB (also referred to as ACREB), or Wnt inhibitory factor (Wif). PCBs or vehicle (DMSO at 1:1000 dilution) was added to the culture medium for 48 hr beginning at 7 DIV; in a subset of cultures, a CaMK kinase inhibitor (STO-609, 5 μM) or a MEK inhibitor (U0126, 10 μM) was also added to the medium during the same period.
Organotypic hippocampal slices from postnatal day–5 rats were cultured for 3 days as described previously (Lein et al. 2011
). At 3 DIV, slice cultures were biolistically transfected with plasmid-encoding tomato fluorescent protein (TFP) using the Helios gene gun (Bio-Rad, Hercules, CA) per the manufacturer’s directions. A subset of slice cultures was simultaneously transfected with siRNA (small interfering RNA) specific for RyR1 or RyR2. Slice cultures were exposed to vehicle, and PCBs were added to the culture medium during 4–6 DIV. A subset of cultures was also exposed to FLA365 [4-(2-aminopropyl)-3,5-dichloro-N
-dimethylaniline] (10 μM), which was added to the culture medium during the same period.
Dendritic morphology was quantified from digital images of green fluorescent protein–positive (GFP+
) or TFP+
neurons using Image J version 1.44p with the Neuron J plug-in version 1.42 to trace neurons (Meijering et al. 2004
Calcium imaging. Spontaneous and electrically evoked Ca2+ transients were measured in dissociated hippocampal neurons cultured on Greiner CELLSTAR® micro-clear wells (Sigma-Aldrich, St. Louis, MO). Cells were loaded with the Ca2+-sensitive dye Fluo-4 AM (5 µM; Invitrogen) at 37°C for 30 min in imaging buffer consisting of 140 mM sodium choride (NaCl), 5 mM potassium chloride (KCl), 2 mM magnesium chloride (MgCl2), 2 mM calcium chloride (CaCl2), 10 mM HEPES, and 10 mM glucose, at pH 7.4, and supplemented with 0.05% BSA (bovine serum albumin). Cultures were washed three times with imaging buffer and transferred to the stage of an inverted Olympus IX70 microscope (Olympus America, Center Valley, PA) equipped with a 60 × 1.25 numeric aperture objective. Fluo-4 was excited at 494 nm using a DeltaRam illuminator (Photon Technologies Int’l., Birmingham, NJ); fluorescence emission was captured at 510 nm. Full-frame images were captured with an Evolve® cooled charge coupled device camera (Photometrics, Tucson, AZ) at 30 frames/sec (fps) using EasyRatioPro software (Photon Technologies Int’l.). In a subset of experiments, cultures were exposed to PCB-95 (2,2´,3,5´6-pentachlorobiphenyl; 2, 20, or 200 nM) from 7–9 DIV before loading with Fluo-4. After baseline recording, cultures were sequentially stimulated with electrical bipolar field pulses (0.5 millisec) at 1, 2.5, 5, and 10 Hz for 10 sec with 50-sec interstimulus rest periods using platinum electrodes connected to a Master 8 stimulator (A.M.P.I, Jerusalem, Israel). After acquisition, regions of interest were drawn freehand to encompass soma and distal dendrites (separated from the soma by a length of > 2 times the soma diameter). Movies were replayed to quantitatively measure changes in Fluo-4 fluorescence within the regions of interest.
At 7 DIV in separate experiments, spontaneous synchronized Ca2+
transients were measured from hippocampal neurons that had not been previously exposed to PCB-95 before loading with Fluo-A. PCB-95 (200 nM) or vehicle (DMSO at 1:1,000 dilution) in imaging buffer was acutely introduced into cultures by continuous flow perfusion (~ 1 mL/min). In a subset of cultures, ryanodine (500 µM; EMD Biosciences, Philadelphia, PA) was added to the culture medium 1 hr before loading the cells with Fluo-A to irreversibly block RyR channel activity (Buck et al. 1992
; Zimanyi et al. 1992
), which was verified by a challenge with 4-chloro-m
-cresol (4CmC, 100 µM; Sigma-Aldrich). Changes in cytoplasmic Ca2+
were continuously recorded at 30 fps. Ca2+
transients > 2 times the baseline amplitude were scored as oscillations. The number of oscillations was compared between vehicle and PCB-95–treated neurons. Transient amplitude was measured by normalizing peak change in Fluo-A fluorescence (ΔF) to the fluorescence baseline (F0
) and presented as mean ΔF/F0
for each neuron included in the analysis. Statistical comparisons were made using neurons (n
= 30) obtained from three separate dissections. Statistical analysis was performed using unpaired Student’s t
Quantitative polymerase chain reaction (qPCR).
Total RNA was isolated from dissociated hippocampal neuron cultures (9 DIV) using Trizol (Invitrogen) according to manufacturer’s instructions. Levels of Wnt2
mRNA were quantified by qPCR and normalized to GAPDH
(glyceraldehyde 3-phosphate dehydrogenase gene) mRNA levels in the same sample. Primer sequences and a more detailed description are provided in Supplemental Material, p. 4