lumbar enlargement were made with a camera lucida attachment under 
dark-field illumination; then under bright-field conditions the distribution of 
Fos-positive cells was plotted with a 4X objective and a camera lucida. 
To quantitate the anatomical results, the spinal cord drawings were divided 
into four segments: (1) the superficial laminae (laminae I, llo, and Ili), (2) the 
nucleus proprius (laminae III and IV), (3) the base of the dorsal horn (laminae 
V and VI), and (4) the intermediate zone and the ventral horn (laminae VII, 
VIII, IX, and X). The number of Fos-positive cells in each of these regions was 
counted and averaged so that each animal had a mean value for regional Fos 
expression. In addition, the sum of the mean regional values (i.e., superficial 
Fos+nucleus proprius Fos+neck Fos+ventral Fos) was used as a measure 
of the total number of Fos-immunoreactive neurons in each animal. The 
investigator responsible for plotting and counting the Fos neurons had no 
knowledge of the drug treatment of each animal. 
In light of the controversy concerning the factors that induce Fos expression, 
a significant finding using the formalin stimulus is that the evoked Fos 
immunoreactivity is largely found ipsilateral to the stimulated hindpaw 
(figure 1). This finding reinforced the idea that the increased number of 
Fos-positive cells was not related to handling or stressing of the animal during 
the protocol. In addition, the rostral-caudal distribution of the formalin-evoked 
Fos immunoreactivity was restricted to the lumbar cord (figure 2), with maximal 
staining in the lumbar enlargement (L4-L5), the area that receives the densest 
primary afferent innervation from the portion of the sciatic nerve territory at the 
site of hindpaw injection. The greatest number of Fos-immunoreactive neurons 
was seen in the superficial laminae of the cord (laminae I and llo), in the neck 
of the cord (lamina V and VI), and in lamina VII and around the central canal. 
Each of these areas has previously been shown to contribute to the processing 
of nociceptive information in the cord (Besson and Chaouch 1987). 
The time course of the Fos expression also closely mirrored the time 
course of the behavioral response (figure 3). Thus, the maximal levels of 
Fos immunoreactivity were seen at 1 to 2 hours post-formalin injection, with 
decreasing activity over the next 6 hours; by 24 hours, almost no Fos staining 
remained in the cord. Taken together with the finding that the level of basal 
Fos expression in the cord is low, these results led to the conclusion that the 
Fos immunoreactivity in the cord is related to the noxious nature of the formalin 
stimulus and not to nonspecific factors secondary to the animal’s perception of 
the stimulus (e.g., stress). 
FOS EXPRESSION AFTER VISCERAL NOXIOUS STIMULATION 
Further evidence in support of this hypothesis is derived from studies of the 
pattern of Fos immunoreactivity evoked by a noxious visceral stimulus 
137 
