Isolation and culture of neurons from the adult mouse myenteric plexus

Myenteric neurons and glia were isolated as described recently⁴¹. Briefly, after euthanizing mice, the ileum was immediately removed and placed in ice-cold Krebs solution (118 mM NaCl, 4.6 mM KCl, 1.3 mM NaH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 11 mM glucose and 2.5 mM CaCl₂) bubbled with carbogen (95% O₂/5% CO₂). The ileum contents were discarded by passing Krebs solution using a syringe. The ileum was then divided into short segments which were threaded longitudinally on a plastic rod through the lumen and the longitudinal muscle containing the myenteric plexus (LMMP) strips were obtained using a cotton-tipped applicator. LMMP strips were rinsed three times in 1 ml Krebs and gathered by centrifugation (350 × g, 30 sec). LMMP strips were then minced with scissors and digested in 1.3 mg/ml collagenase type II (Worthington) and 0.3 mg/ml bovine serum albumin in bubbled Krebs (37 °C) for 1 h, followed by 0.05% trypsin for 7 min. Following each digestion, cells were triturated and collected by centrifuge (350 x g for 8 min). Cells were then plated on laminin (BD Biosciences) and poly-D-lysine coated coverslips in Neurobasal A media containing B-27 supplement, 1% fetal bovine serum, 10 ng/ml glial cell line-derived neurotrophic factor (GDNF, Neuromics, Edina, MN), and penicillin/streptomycin. Half of the cell media was replaced every 2–3 days with fresh complete neuron media.

All chemicals and reagents were obtained from Sigma Aldrich (St Louis, MO), unless otherwise noted, except cell culture reagents, which were purchased from Gibco (Grand Island, NY) and the HIV Tat_1–86 protein (Immunodiagnostics, Inc.). Male Swiss Webster mice (25–30 g, Harlan Sprague Dawley, Inc.) or male and female doxycycline (DOX)-inducible, HIV-Tat_1–86 transgenic mice (25–30 g) were used. The HIV-Tat_1–86 transgenic mouse model was developed on a C57BL/6J hybrid background and is described in detail elsewhere⁴² (17). Tat expression, which is under the control of a tetracycline responsive, glial fibrillary acidic protein (GFAP)-selective promoter, was induced with a specially formulated chow containing 6 mg/g DOX (Product#: TD.09282; Harlan, Indianapolis, IN, USA), fed to both the Tat− controls and the inducible Tat+ mice. Toll like receptor 4 (TLR4) knockout mice were used to determine the role of TLR4 in the interaction of LPS and Tat. Mice were euthanized by cervical dislocation.