Expression of c‐fos protein in interneurons and projection neurons of the rat spinal cord in response to noxious somatic, articular, and visceral stimulation
Authors: Menétrey D. , Gannon A. , Levine J. D. , Basbaum Allan I. 
Published: January 2005

This study used immunocytochemistry to examine the pattern of noxious‐stimulus evoked expression of the proto‐oncogene c‐fos in the spinal cord of the rat. Both noxious somatic and joint stimulation in awake rats evoked the expression of c‐fos protein in similar areas of the lumbar spinal cord. C‐fos‐immunoreactive neurons were found in laminae I and outer II, in the lateral part of the neck of the dorsal horn, and in laminae VII, VIII, and X. All of the labelled neurons were located ipsilateral to the injured hindpaw, except for lamina VIII where bilateral labelling was recorded. The c‐fos‐immunoreactive neurons in lamina I extended from the L3 segment to the rostral sacral cord; staining in outer lamina II was only found at the L4 segment. The more deeply located cells, of the dorsal and medioventral horns, had the most extensive rostrocaudal spread; they were found from L1 through the rostral sacral segments.

The pattern of c‐fos‐immunoreactivity produced by visceral stimulation, in anesthetized rats, differed in several ways from that produced by somatic stimulation. First, there was considerable bilateral, symmetrical labelling of cells. Second, there was a much more extensive rostrocaudal spread of the labelling, from cervical through sacral cord. Third, the greatest rostrocaudal spread was found for neurons in the superficial dorsal horn; labelled cells in the neck of the dorsal horn and in lamina X were restricted to segments at the thoracolumbar junction, which is also where the superficial dorsal horn cells were most concentrated. Fourth, there were very few labelled neurons in the outer part of the substantia gelatinosa.

To determine whether any neurons that express the c‐fos protein in response to noxious stimulation project to supraspinal sites, we combined the immunocytochemical localization of c‐fos with the localization of a retrogradely transported protein‐gold complex that was injected into the thalamic and brainstem targets of the major ascending spinal pathways. In rats that received the somatic noxious stimulus, 90% of all of the c‐fos projection neurons were recorded in four major areas of the cord: lamina I (37%), the lateral part of the neck of the dorsal horn (24%), laminae VIII (9%), and X (29%). The remainder were scattered throughout the spinal gray. With the exception of lamina VIII, which contained c‐fos projection neurons contralateral to the inflamed paw, all of the c‐fos projection neurons were located ipsilateral to the injured paw. Although c‐fos‐immunoreactive neurons and retrogradely labelled cells were found in many other areas of the spinal gray that contain nociresponsive neurons, few were double‐labelled. Finally, retrogradely labelled cells that expressed c‐fos in response to visceral stimulation were only found in the superficial dorsal horn. They were distributed from cervical through sacral levels; most were at the thoracolumbar junction.

This study demonstrates that the c‐fos protein can be used as a functional marker to identify the spinal neurons that are activated by different forms of noxious stimulation and indicate that in the awake, freely moving animal, activity in projection neurons of four regions, lamina I, the lateral neck of the dorsal horn, laminae VIII and X, contribute to the central transmission of nociceptive messages that are probably involved in the conscious appreciation of pain.