In response to the letter to the editor by Dr. Hagihira regarding our manuscript entitled “Nitrous Oxide Activates GABAergic Neurons in the Spinal Cord in Fischer Rats,” we offer the following point-by-point response.
First, Hagihira writes that neither he 1nor Sun et al. 2found “increase of c-Fos expression by inhalation of nitrous oxide.” However, neither examined the effect of nitrous oxide alone on c-Fos expression in the spinal cord as we did in our study. Also, their experimental designs were not capable of eliciting the antinociceptive effect of nitrous oxide. Hagihira et al. 1administered nitrous oxide after formalin injection, although it takes 15–30 min for nitrous oxide to show a significant antinociceptive effect. In the study by Sun et al. , 2nitrous oxide was administered 20 min before formalin injection but was discontinued 5 min after formalin injection, although noxious stimuli by formalin last at least 1 h. Therefore, it is understandable why both studies failed to show a significant effect of nitrous oxide on formalin-induced nociceptive behavior and c-Fos expression.
Second, Hagihira comments that “… neurons exist in laminae III and IV receive only proprioceptive inputs and do not involve noxious input processing,” which we do not contest. Our finding that nitrous oxide induces c-Fos expression in laminae III and IV are consistent with this fact because it is the γ-aminobutyric acid–mediated interneurons that are affected by nitrous oxide and not the primary afferent neurons conveying nociceptive input. Hagihira cites two studies (one published by Westlund et al. 3in 1983 and another by himself 4in 1990) to substantiate his statement that “… there are many noradrenergic terminals in laminae I and II but few in laminae III and IV.” However, he ignores many other studies that have shown that noradrenergic neurons are widely distributed within the entire spinal cord, including the motor neurons (e.g. , Commissiong 5, Aramant et al. 6, Rajaofetra et al. 7).
Third, Hagihira is concerned about the high “background” c-Fos expression in the spinal cord of our study (i.e. , approximately 40 cells per section), higher than that seen in previous studies (Hunt et al. 8in 1987, Menétrey et al. 9in 1989, and Bullitt 10in 1990). We can only comment that the sensitivity of antibodies and imaging have increased over this period; support for this contention is the fact that in the report by Hunt et al. 8in 1987, there was considerably less c-Fos induction by noxious stimuli. Unlike other investigators, we have not confined our examination only to the dorsal horn of one half of the spinal cord but have examined the entire gray matter of both halves of the spinal cord, which results in a higher number of c-Fos–positive cells in the control samples.