Mouse current vocalization threshold measured with a neurospecific nociception assay: the effect of sex, morphine, and isoflurane

Abstract

Sine-wave electrical stimulation at frequencies 2000, 250, and 5Hz to respectively evaluate Aβ, Aδ, and C sensory neurons has recently been added to the armamentarium used to evaluate sensory neurons. We developed an automated nociception assay using sine-wave stimulation methodology to determine current vocalization threshold in response to 2000, 250, and 5Hz and examine the effects of sex, analgesics, and anesthetics in mice. At baseline, males had significantly higher mean current vocalization thresholds compared with female mice at 2000, 250, and 5Hz (p≤0.019). By 1h after intrathecal injections of morphine there were significant increases in current vocalization threshold percent changes from baseline that varied with doses (p=0.0001) and frequency used (p<0.0001). Specifically, with increasing doses of morphine, there were significantly greater increases in current vocalization threshold percent changes from baseline in response to 5Hz compared with 250 and 2000Hz stimulation in a significantly ordered pattern: 5Hz>250Hz (p<0.0001) and 250Hz>2000Hz (p=0.0002). Forty-five minutes after exposure, there were no effects of isoflurane on current vocalization thresholds at any frequency. Therefore, our findings suggest that this automated nociception assay using sine-wave stimulation in mice, can be valuable for measurements of the effects of sex, opioids, and anesthetics on the response to electrical stimuli that preferentially stimulate Aβ, Aδ, and C-sensory fibers in vivo. This investigation suggests the validation of this assay and supports its use to examine mechanisms of nociception in mice.

Fig. 1

Components of the nociception assay used to study the effects of sex, opioids and anesthetics in current vocalization threshold to electrical stimuli. The delivery of stimulation and detection of nocifensive behavior recognition is entirely automated.

Fig. 2

Experimental design and time course of the study. Animals participating in the morphine study received an intrathecal injection of morphine at the time indicated and those in the isoflurane study did not.

Fig. 3

Mean ± SEM baseline current vocalization thresholds in male (gray bars) and female (black bars) C57BL6/J and B6129Sf\J mice in response to 5 (Panels A and D), 250 (Panels B and E), and 2000 (Panels C and F) Hz. * indicates p ≤ 0.01 for male versus female comparisons.

Fig. 4

Effect of sex on effect of intrathecal morphine. Panels A and C show least square mean ± SEM current vocalization threshold percent changes from baseline in male and Panels B and D in female C57BL6/J mice at 1 h (Panels A and B) and 3 h (Panels C and D) after administration of escalating doses of intrathecal morphine. Percent changes from baseline after intrathecal morphine were similar comparing male versus female mice (all p > 0.26).

Fig. 5

Effect of intrathecal morphine expressed as current vocalization threshold percent changes from baseline at 1 (Panel A) and 3 h (Panel B) in response to 5, 250, and 2000 Hz sine-wave stimulation. * represents significant (p < 0.05) percent changes from baseline for 5 Hz stimulation, † (p < 0.05) for 250 Hz, and ‡ (p < 0.05) for 2000 Hz. Open and filled symbols represent least square means current vocalization threshold percent changes from baseline for male and female mice combined (as there was no significant effect of sex) and bars represent the standard error for 5, 250, and 2000 Hz. With increasing doses of morphine, percent changes in current vocalization threshold increased and were significantly greater for 5 Hz compared with 250 and 2000 Hz (Panel A). Current vocalization threshold percent changes from baseline at 1 h (Panel A) were significantly ordered: 5 Hz > 250 Hz (p < 0.0001) and 250 Hz > 2000 Hz (p = 0.0002). The curves represent polynomial smooth regression lines for 5, 250, and 2000 Hz frequencies stimulation as indicated in the legend. Data were modeled using repeated measures to compare the effect of increasing doses of morphine among the 3 frequencies at different doses.