2. What is c-Fos?
Tissue-Specific Gene Expression Pattern of the Fos Gene
(Genomics Institute of the Novartis Research Foundation,
GNF)
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NFAT(green), Jun(blue) and Fos(red),
bound to DNA
c-Fos is rapidly and transiently expressed in neurons in response to stimulation
3. c-Fos as an Indirect Marker of Functional Activity
• Studies show that various types of noxious stimulation (thermal, mechanical,
chemical) induce c-Fos expression in the brain and spinal cord.
• “Induction of c-Fos-like protein in spinal cord neurons following sensory
stimulation”, Hunt et al. 1987.
– Noxious stimulation (mustard oil or radiant heat) to the hind paw resulted
in a massive increase in the expression of Fos in neurons in the dorsal
horn of the lumbar spinal cord.
• Nociception-induced c-Fos expression in spinal neurons is suppressed by
administration of analgesic drugs
– Acclimatizing to environment conducted to reduce Fos expression due to
handling, restraint, or novel environment stressors
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4. Increasing Interest in the Scientific Community
4
The number of publications each year from 1995 to 2015 identified by a
literature search on the ScienceDirect database using the keyword “fos”
combined with either “pain”, “nociception”, “nociceptive”, or “noxious”.
5. Implementing a Translational Tool
Advantages of using c-fos expression as a tool for
evaluating neural basis of nociception:
– Easy to identify neuronal populations that
respond to noxious stimulation
– c-fos expression can be analyzed quantitatively
(counting of neurons immunoreactively
labeled for Fos)
– Provides reliable basis for comparison of
various manipulations on nociceptive
processing
• Basal c-fos expression is low in most
neurons
– Does not require the use of anesthesia during
noxious stimulation
• nociceptive processing under normal
conditions
– Immuno-labeling of Fos protein with other
immunocytochemical & tract-tracing
procedures (ex. retrograde labeling)
• identify c-fos-expressing neuronal
projections
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Photomicrograph of in-vivo basal c-Fos
expression in the rat spinal cord. Insets are
magnified.
Figure 4A, "Sympathetic-correlated C-Fos Expression in the
Neonatal Rat Spinal Cord in Vitro." Open-i. API, 2009. Web.
17 Aug. 2015.
6. Project Overview
Development of immunohistochemical detection of Fos
protein after noxious stimuli and alterations in c-Fos
expression with the addition of pain-relieving drugs
morphine and pregabalin.
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7. Materials & Methods: AFP Study
• Unbiased, automated measurement of
response to formalin insult
• Metal bands placed on left hind paw &
rats acclimated to the test chamber
(approx.15 min)
• Test article administered (PO, IP, SC, or
IV)
• 50 uL formalin solution administered
into dorsal surface of paw
• Rat placed in test chamber- number of
paw movements recorded for up to 60
minutes post-formalin injection
• Rat euthanized 1hr after study
conclusion
– Previous literature shows c-fos
expression peak ~2hr post-formalin
injection
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8. Results: AFP Study Data
–First (acute) phase: 0-9 minutes; results from direct chemical activation (i.e. formalin) of myelinated and
unmyelinated nociceptive afferent fibers- acute mechanical pain.
–Short quiescent period
–Second (tonic) phase: 10-60 minutes; characterized by persistent shaking or licking of the injected paw due to
inflammatory pain and central sensitization.
These biphasic behavioral responses are paralleled by a biphasic discharge of DH nociresponsive
neurons.
8
16. Columnar Representation of Fos-like
Immunoreactivity Expression
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Effects of SC (subcutaneous) morphine and PO (per oral) pregabalin on formalin-
induced c-fos expression
17. Relationship between Nociceptive Behavior
and c-Fos Expression
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Effects of SC (subcutaneous) morphine and
PO (per oral) pregabalin on formalin-
induced nociceptive behavior
Effects of SC (subcutaneous) morphine and
PO (per oral) pregabalin on formalin-induced
c-fos expression
18. Summary & Conclusions
Immunohistochemical techniques were optimized for c-Fos expression and
can be successfully applied to future studies.
There is no relevant literature on the effects of pregabalin on c-Fos expression
in the AFP model. There is literature demonstrating c-Fos expression
reduction with pregabalin administration in neuropathic pain models. This is a
novel finding.
Future studies can be conducted to profile various classes of analgesics to
determine the signature of each on noxious stimulus-evoked FLI.
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20. References
• "Sympathetic-correlated C-Fos Expression in the Neonatal Rat Spinal Cord in Vitro."
Open-i. API, 2009. Web. 17 Aug. 2015.
• Krukoff, Teresa L. "C-fos Expression as a Marker of Functional Activity in the Brain."
Neuromethods 33 (1999): 213-30. Print.
• Hunt, S. P.; Pini, A.; Evan, G. Induction of c-fos-like protein in spinal cord neurons
following sensory stimulation. Nature 328:632–634; 1987.
• Hahm, T. S., H. J. Ahn, S. Ryu, M. S. Gwak, S. J. Choi, J. K. Kim, and J. M. Yu. "Combined
Carbamazepine and Pregabalin Therapy in a Rat Model of Neuropathic Pain." British
Journal of Anaesthesia 109.6 (2012): 968-74. Web. 18 Aug. 2015.
• Harris, Justin A. "Using C-fos as a Neural Marker of Pain." Brain Research Bulletin 45
(1997): n. pag. Elsevier. Web. 17 Aug. 2015.
• Gogas, K. R. "The Antinociceptive Action of Supraspinal Opioids Results from an
Increase in Descending Inhibitory Control." Neuroscience 42.3 (1991): n. pag. Print.
• Jasmin, L. "Differential Effects of Morphine on Noxious Stimulus-evoked Fos-like
Immunoreactivity in Subpopulations in Spinoparabrachial Neurons." The Journal of
Neuroscience (1994): 7252-260. Print.
• Presley, R. W.; Mene´trey, D.; Levine, J. D.; Basbaum, A. I. Systemic morphine
suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal
cord. J. Neurosci. 10:323–335; 1990.
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