Picopump

Neurons within brain slices were visualized with infrared or visible differential interference contrast (DIC) optics. Neurons in the ventral inferior (VI) aspect of the MHb were targeted for recordings, as previously described (Shih et al., 2014 (link), 2015 (link)). Electrophysiology experiments were conducted using a Scientifica SliceScope upright microscope. A computer running pCLAMP 10 software was used to acquire whole-cell recordings along with an Axopatch 200B amplifier and an A/D converter (Digidata 1440A). pClamp software and acquisition hardware were from Molecular Devices. Data were sampled at 10 kHz and low-pass filtered at 1 kHz. Immediately prior to gigaseal formation, the junction potential between the patch pipette and the superfusion medium was nulled. Series resistance was uncompensated.
To record physiological events following local application of drugs, a drug-filled pipette was moved to within 20–40 μm of the recorded neuron using a second micromanipulator. The drug (dissolved in recording solution) was dispensed onto the recorded neuron by using a Picopump (World Precision Instruments) at an ejection pressure of 12 psi for 250 ms. The ejection volume varied depending on the goal of the experiment. Atropine (1 μM) was present in the superfusion medium when using ACh application to prevent activation of muscarinic AChRs.

All surgical and postoperative care procedures were performed in accordance with The Ohio State University Institutional Animal Care and Use Committee. Adult female Sprague-Dawley rats (~250 grams; n = 56) were randomly assigned to treatment groups (vehicle control, iron, iron+LPS, LPS; n = 10/group), then anesthetized with an intra-peritoneal injection of ketamine (80mg/kg) and xylazine (10mg/kg). Using aseptic technique, a laminectomy was performed at the T8 vertebral level. Custom pulled UV-sterilized glass micropipettes beveled to an outer tip diameter of 25–40μm were loaded with the proper solution and positioned 0.7mm lateral to the dorsal spinal cord midline. Using a hydraulic micropositioner (David Kopf Instruments, Tujunga, CA), pipettes were lowered 1.1mm into the spinal cord. For histological experiments, a 500nl bolus injection was administered to the lateral gray-white matter border using a PicoPump (World Precision Instruments). For tissue RNA experiments, a 200nl bolus injection was administered bilaterally in the lateral gray-white matter border. Injection sites were labeled with sterile charcoal (Sigma), muscles surrounding the laminectomy were sutured, skin was stapled with wound clips, and rats were given 5cc sterile saline (subcutaneous) before being placed into a warmed recovery cage. Two rats died due to complications with anesthesia.