Electrical stimulation of the whisker
pad and forepaw was described in our previous study (Yu et al., selleck chemicals llc 2010 and Berwick et al., 2004). For the forepaw stimulation, two needle electrodes were inserted between digits 1, 2 and 3, 4. For whisker pad stimulation, an electrode pad with five pins (one cathode in the center of a 5 × 5 mm square with four anodes at each corner) was designed. To reduce the cross-subject variation, the center pin was positioned at the third whisker of the caudal side of row C for each rat. An isolated stimulator (WPI, FL) supplied 330 μs pulses repeated at 3 Hz to the whisker pad and forepaw simultaneously upon demand at varying amount. The electrical LBH589 current was set from 1.0 to 3.0 mA with 0.5 mA increments to the whisker pad. It is noteworthy that electrical stimulation at 2.5–3.0 mA led to the subcortical BOLD-fMRI responses in the ipsilateral thalamic area and habenular nuclei, which could be related to pain processing at the high stimulation intensities (Hikosaka, 2010).
A detailed procedure was described previously (Tucciarone et al., 2009). For thalamic injections, rats received 250 nl of 50 mM MnCl2 solution (0.9% saline) into the left hemisphere (Bregma −3.0, lateral − 3.0, and ventral 5.7 mm), contralateral to the intact whisker pad of IO rats. The stereotactic coordinates were determined according to the Paxinos and Watson (2007) atlas. Animals were anesthetized by isoflurane. A small bur hole was drilled after exposing the skull. A homemade glass injection needle was placed at the proper coordinates in the stereotactic frame. Injections were about performed slowly over 5–6 min using a microinjector (Narishige, Tokyo), and the needle was slowly removed after being kept into the injection site for 10 min. MRI was performed right after stereotactic injections to make sure MnCl2 delivered to the proper site and
at 4 to 6 hr post injection to analyze Mn in the cortical lamina (Tucciarone et al., 2009). For MRI scans, rats were anaesthetized with 1%–2% isoflurane using a nose cone and rectal temperature was maintained at 37°C ± 1°C by a heated water bath. After surgery and in between scans, the rats were allowed to recover and were free to roam within their cages. No abnormalities were observed after injection in all rats. All images were acquired with an 11.7 T/31 cm horizontal bore magnet (Magnex, Abingdon, UK), interfaced to an AVANCE III console (Bruker, Germany), and equipped with a 12 cm gradient set, capable of providing 100 G/cm with a rise time of 150 μs (Resonance Research, MA). A custom-built 9 cm diameter quadrature transmitter coil was attached to the gradient. A 1 cm diameter surface receive coil with transmitting/receiving decoupling device was used during imaging acquisition.