2.5. Dorsal horn electrophysiology

In vivo extracellular single-unit recordings were performed as previously described.³⁷ Rats were anaesthetised with isoflurane (induction 5% in O₂). The animals were tracheotomised; air flow and breathing rate were adjusted to animal weight with a small animal ventilator (Model 687; Harvard Apparatus, MA). Procedures were performed under constant isoflurane anaesthesia (maintenance 1.8% in O₂, Univentor Anaesthesia Unit 400; Royem Scientific, Luton, United Kingdom). A heating blanket with feedback control was used to maintain body temperature throughout recording (Model 507220F; Harvard Apparatus). The rat was mounted on a stereotactic frame (Kopf Instruments, Tujunga, CA) and a laminectomy performed to expose the lumbar spinal cord. The vertebral column was secured with a clamp rostral to the laminectomized area, the dura and pia mater were removed, and the exposed spinal cord was covered with mineral oil.

A 6-μm tipped glass-coated carbon fibre electrode (Kation Scientific, Minneapolis, MN) was lowered through the spinal cord in 2 to 10 μm steps with a Microdrive (Scientifica Microdrive, Scientifica, Uckfield, United Kingdom). To isolate individual cells, the plantar surface of the hind paw was stroked as search stimulus for dorsal horn wide-dynamic-range (WDR) neurons in laminae IV-VI, at a depth between 400 and 800 μm. Stimulus-evoked potentials were digitalized by a Powerlab interface and recorded using Labchart software (AD Instruments Ltd, Oxford, United Kingdom). The cutaneous receptive field (RF) on the plantar skin to tactile (a camel hair brush) and noxious pinch stimulation (fine forceps) was mapped using InkScape software. Spontaneous activity in the absence of cutaneous stimulation was recorded for 1 minute. The number of spikes evoked during a single 0.5-second brush (repeated 3 times at >1 second intervals), a single 1.5-second pinch (repeated 3 times at >1 second intervals), and a single 0.5-second vFh stimulus (range: 1.202; 2.041; 3.63; 5.495; 8.511; 15.136; and 28.84 g; 3 applications to peak RF, >1 second interstimulus interval) was recorded, with a minimum 10-second interval between stimuli. A total of 313 cells were recorded from the ipsilateral and contralateral dorsal horn of 41 animals (206 ipsilateral and 107 contralateral). Outliers were removed and 298 cells (191 ipsilateral and 107 contralateral) were included in the analysis, summarized in Table ​Table2.2. The depth of cells recorded and analysed per group is included in Table ​Table33.

Number of wide-dynamic-range cells (ipsilateral and contralateral) recorded in each group.

Depth of cells (μm, wide-dynamic-range neurons) recorded in the spinal dorsal horn of each group.