C-
fos encodes a nuclear protein that regulates the transcription of other target genes and of its one promoter. Its detection within the brainstem and spinal cord neurons is the best studied technique to map the postsynaptic nociceptive pathways in numerous cephalic pain models [
5,
18,
34]. In the field of migraine research fos protein immunoreactivity offers a method to identify subpopulations of neurons activated in response to noxious stimuli and identify related nociceptive pathways [
5]. The majority of studies have employed this technique to map neuronal activation not only throughout the trigeminovascular system [
35‐
39], but also higher structures involved in the ascending and descending modulatory control of pain [
6,
40‐
42], thus, greatly enhancing our understanding of the pathophysiology of migraine. As with other models of the components of migraine the use of fos expression has certain limitations [
43]. The major one being the model can only be as good as the stimulus since it is the stimulus that drives the expression of fos protein. Several intracranial structures have been stimulated chemically or electrically to induce fos within trigeminal nucleus caudalis, including the meninges [
18,
20,
22,
23], trigeminal ganglion [
44], the superior sagittal sinus [
35] and middle meningeal artery [
45]. It seems that the activation of specific structures of the trigeminovascular system (e.g. the superior sagittal sinus) leads to more clinically relevant conclusions [
35,
46]. It is noteworthy that induction of fos to quantifiable levels requires a strong consistent stimulation that is often not physiological [
47,
48]. It must also be remembered that
c-
fos is not expressed in all neurons as with the dorsal root ganglion cells [
34], thus, lack of fos protein expression does not equate to lack of neuronal activity. Technical difficulties in inducing
c-
fos expression within trigeminal nucleus caudalis are also important [
49]. One final limitation of the model is seen via direct activation of the antinociceptive descending pathways, which elicits
c-
fos expression in spinal neurons even in the absence of any nociceptive input [
50].