sensory neurons, these findings provide a potential biological mechanism for some of the abnormalities in pain sensation seen in patients with schizophrenia. Materials and Methods Animals All experiments were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and studies were approved by Institutional Animal Care and Use Committees at Columbia University (#3132) and Stony Brook University (#1618 and #1792). Mice heterozygous for an isoform specific disruption of Type III Nrg1 (Nrg1tm1Lwr) and their wild-type littermates were generated and genotyped as previously described [7]. Animal lines were back-crossed onto a C57/Bl6 background. WT and Type III Nrg1 heterozygous (Type III Nrg1+/2) littermates were generated from heterozygote crosses. TRPV12/2 mice were obtained from Jackson Labs. As TRPV12/2 mice are viable and fertile, TRPV12/2 crosses were used to generate the TRPV12/2 mice used in experiments. Only male mice were used for experiments unless specifically stated. Animals were housed 2–4 per cage, maintained on a 12 h light/dark cycle and provided with food and water ad libitum. Cell culture Primary DRG cultures were prepared from P21 WT or Type III Nrg1+/2 male mice. Lumbar DRGs were dissected into L-15 media (Sigma) and incubated in a 0.1% collagenase (Type IV, Worthington) solution for 1 hour at 37uC/5% CO2. Cells were rinsed once in L-15 before being dispersed in MEM (Gibco) supplemented with 10% Fetal Bovine Serum (Gemini Bio- Products), 50 u/ml each of penicillin and streptomycin (Gibco), 2 mM L-glutamine (Gibco) and 50 ng/ml NGF (Harlan Biosci- ence) using a series of fire-polished pipettes. For immunostaining and calcium imaging, cells were plated onto 12-mm nitric- acid washed, poly-D-lysine (1 mg/ml, Chemicon) and laminin (100 mg/ml, Sigma) coated glass coverslips (Warner Instruments) at a density of ,4,000 cells/well. Cells were left at 37uC/5% CO2 for 36–48 hours before use. For immunoblots, cells were plated onto poly-D-lysine and laminin coated cell culture plates (6-well plate: 20,000 cells/well; 12-well plate: 10,000 cells/well) and left at 37uC/5% CO2 for 48 hours before use. Reagents Where indicated, the following reagents were applied to cell cultures: Capsaicin (Sigma; prepared as a 100 mM stock solution in 10% ethanol), capsazapine (Tocris Bioscience; prepared as a 10 mM stock in 70% EtOH), 300 ng/ml soluble ErbB4-ECD (sErbB4-ECD) (Bao et al. 2003), 2.5S nerve growth factor (NGF; BT-5025 Harlan Bioproducts), 20 nM wortmannin (WM; Cal- biochem), 50 mM LY294002 (Cell Signaling Technology) and 1 mM PD158780 (Calbiochem). Behavior Male WT and Type III Nrg1+/2 littermates, 5–10 months of age, were used for behavioral tests. The experimenter was blind to the genotype of the mice during testing. Hargreaves Paw Withdrawal Assay Heat pain threshold of the plantar hindpaw was assessed using the Hargreaves method [24]. Animals were placed in pairs in plastic enclosures on an elevated glass plate pre-warmed to 30uC and allowed to acclimate for 1.5 hours. A focused beam of light with the percent intensity set to 15 (IITC Life Science) was used to Type III Nrg1, TRPV1 and Pain Sensation heat the plantar surface of each hindpaw, and the time for the animals to withdraw their hindpaws from the light source was recorded to the hundredth of a second as the reaction latency. For baseline reaction latency measurements each paw was tested 4 separate times on 2 separate days and the values for each paw were averaged together by animal. Averages for reaction latency were compared by genotype. Cold Plate Test Noxious cold sensitivity of the plantar surface of the hindpaw was assessed using a cold plate analgesia meter (IITC Life Science). To measure pain, mice were placed on the metal surface pre-set to 0uC, and the latency to generate a nocifensive response (lifting or flicking the hindpaws or jumping), as well as the total number of nocifensive responses generated within the five-minute interval in which the animal remained on the plate, were recorded. Averages for each variable were compared by genotype. von Frey Mechanosensitivity Test Mechanical withdrawal thresholds of the plantar hindpaw were assessed using the von Frey method. Animals were placed on a plastic grid in plastic enclosures and allowed to acclimate for 20 minutes. Filaments of increasing durability (IITC Life Science) were used to apply escalating levels of force to the plantar hindpaw. Each filament was applied 5 times to the hindpaw and the number of times the animal withdrew its paw from that stimulus intensity was recorded and expressed as a percent response. Average percent response at each stimulus intensity was compared by genotype. Modified Hargreaves Paw Withdrawal Assay with Capsaicin Application A modified version of the Hargreaves method was used to assess the development of inflammatory thermal hypersensitivity in the plantar hindpaw following application of capsaicin. The Har- greaves Assay was used as described above, with the following modifications. The glass plate was left at room temperature (25uC) and the percent intensity of the light source was set to 10 (IITC Life Science). Baseline measurements were made testing each paw 4 times and the paw withdrawal latencies were averaged by paw. The following day, animals were lightly anesthetized with 2% isofluorane, and 20 ml of 0.075% capsaicin cream (Zostrix) was applied to the right hindpaw and lightly massaged into the skin. The animals were allowed to recover on the elevated glass plate in plastic enclosures for 20 minutes before reaction latency was again assessed for each paw. Absolute reaction latencies, as well as percent changes from baseline, were compared by genotype. Histology For immunofluorescent or immunohistochemical staining of DRG tissue, adult male mice were deeply anesthetized with a mixture of ketamine-xylazine (90 mg/kg and 10 mg/kg, respec- tively) and transcardially perfused with 0.1 M PBS for 2 minutes followed by 4% paraformaldehyde (PFA) in PBS for 2 minutes. Following perfusion, L4 and L5 DRG were dissected out bilaterally, post-fixed in 4% PFA overnight, embedded in paraffin and cut in 7 mm thick sections on a microtome. Serial sections from litter matched adult male WT and Type III Nrg1+/2 mice were collected on Superfrost plus microscope slides such that each slide contained representative sections taken approximately every 150 mm throughout the entire DRG. To visualize DRG cell profiles, paraffin-embedded tissue was subjected to antigen retrieval, blocked in 10% normal donkey serum (NDS) in PBS PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25108