Ventral and dorsal root activity was recorded via suction electrodes (A-M Systems). Fluorescent dI3 INs were targeted on the basis of their location in the intermediate

spinal cord and recorded with care to not include cells that were deep (motoneurons). Most recordings were from L4 and L5 segments, but, when recording in rostral lumbar segments, neurons near the surface (possible autonomic motoneurons) were also avoided. Sensory fibers have different recruitment thresholds, which depend drug discovery on the size of the fiber and the degree of myelination (Erlanger and Gasser, 1930). To express stimulation strength, we defined T as either the lowest stimulus strength at which DR volleys or CDPs were first seen (if electrodes present) or the strength at which ventral root responses were seen. We classified the responses of dI3 INs as either low-threshold (1–2 T), which would include group I muscle afferents and low-threshold cutaneous afferents (Aβ) but also some group II and Aδ fibers, or high-threshold afferents (5–10 T), which putatively included group III and group IV muscle afferents and unmyelinated C fiber nociceptive afferents. Intermediate stimuli were classified as medium

threshold. T was typically between 4 and 20 μA in vitro. Adult mice were implanted with bipolar electromyography (EMG)-recording electrodes (Pearson et al., 2005; Akay et al., 2006) as well as cuff electrodes to stimulate the tibial and/or selleck screening library sural nerves. Following 1–3 weeks of recovery, nerves were stimulated with the use of single

or pairs of 250 μs pulses for the tibial nerve or with the use of trains of two Histone demethylase to five pulses for the sural nerve at frequencies of 500 Hz with an interval of 2 s between trains. Stimulation strengths used to attempt to elicit reflexes ranged between 75 and 500 μA (mean, 307 ± 135 μA; n = 11) in the control animals and 40 to 750 μA (mean, 248 ± 228 μA; n = 8) in the mutant animals (p = 0.31). In contrast, the nociceptive threshold (producing vocalizations) ranged between 300 and 1,500 μA (mean, 821 ± 356 μA; n = 7) in the control animals and between 250 and 900 μA (mean, 571 ± 216 μA; n = 7) in the mutant animals (p = 0.07). The differences between the stimulation used to elicit the short-latency reflexes and the threshold for vocalization were significant (paired t test, p < 0.05, n = 5 control and 2 dI3OFF animals). We implanted chronic epidural cord dorsum electrodes in four animals to determine stimulation thresholds (n = 2 mutant and 2 controls). The threshold to elicit short-latency cord dorsum responses from sural nerve stimulation was between 100 and 300 μA (Figure S5B; n = 2 mutants and 1 control); i.e., in the same range that we used to elicit reflex responses.