C-Fos

c-Fos Protein

The immediate early gene product c-Fos is commonly used to map neural structures that are activated by the unconditioned and conditioned stimuli in CTA paradigms [Refs]. The c-Fos protein is expressed at a very low constituitive levels in many brain structures. Following US or CS stimulation of the animal, however, transynaptic activation of second messenger cascades causes the rapid but transient synthesis of c-Fos protein within 30 -180min. The c-Fos protein is easily visualized by immunohistochemistry; because c-Fos is a transcription factor, its labeling is discretely localized within cell nuclei. Thus, the presence of c-Fos after stimulation in a central relay implies direct or indirect activation of the relay by the stimulus. Quantification of the number of c-Fos positive cells provides a measure of response magnitude.

c-Fos as a Correlate of CTA Expression

There are only a few scattered reports of neuronal activity correlated with CTA acquisition and expression. Shifts in the electrophysiological response of neurons to a standard gustatory stimulus after CTA acquisition in the NTS (Chang and Scott 1984), PBN (DiLorenzo 1985), amygdala(Yamamoto, Shimura et al. 1991), cortex (Yamamoto 1989), or elsewhere(Brozek, Buresova et al. 1974; Buresova, Aleksanyan et al. 1979) have been reported. Acetylcholine release in the n. accumbens is altered by CTA acquisition (Mark, Rada et al. 1992), and NMDA receptor phosphorylation increases in the gustatory cortex (Rosenblum, Berman et al. 1997). None of these neuronal correlates of CTA expression have been systematically characterized.

We and others have observed(Houpt, Philopena et al. 1994; Swank and Bernstein 1994) that c-Fos expression in the medial intermediate NTS (iNTS) correlates well with CTA expression. Although unconditioned oral infusions of sucrose solution did not induce c-Fos in the iNTS under our conditions, after 3 pairings of sucrose with LiCl injections, intraoral infusion of sucrose produced an aversive behavioral display, and a significant increase in c-Fos expression in iNTS (Houpt, Philopena et al. 1994). LiCl alone, or sucrose after non-contingent pairing with LiCl did not reproduce this c-Fos pattern (Houpt, Philopena et al. 1994). In addition, the induction of c-Fos in the iNTS following CTA expression correlates well with the persistence, forgetting(Houpt, Philopena et al. 1996), and extinction(Houpt, Philopena et al. 1994) of the CTA; it is correlated with CTAs mediated by different tastes and toxins (Swank, Schafe et al. 1995; Houpt, Philopena et al. 1996; Thiele, Roitman et al. 1996); and it is not dependent on aversive behavioral responses (Swank, Schafe et al. 1995; Houpt, Philopena et al. 1996). While the expression of c-Fos is not dependent on abdominal vagal afferent or efferent fibers (Houpt, Berlin et al. 1997), it does require forebrain circuitry (Schafe, Seeley et al. 1995), including the CeA (Schafe and Bernstein 1996) and GC (Schafe and I.L. 1998).

Analysis of c-Fos in CTA

No other neuronal marker of CTA expression has correlated this well with the behavioral expression of a CTA. As a marker of neuronal activity, c-Fos expression has several advantages over other measures of activity:

1. The mechanisms subserving CTA appear to form a distributed system with multiple brain sites activated by conditioned and unconditioned stimuli or generating conditioned responses. c-Fos expression can visualize activity in multiple brain regions of the same animal for the analysis of a distributed network.

2. The pattern of c-Fos expression in the brain provides cellular resolution of neural activity(Sagar, Sharp et al. 1988)that can be quantified by counting the number of labeled cells or measuring the intensity of in situ hybridization. The degree of c-Fos expression can then be correlated with quantifiable behavioral measures.

3. Expression of c-Fos also reveals the nature of well-characterized intracellular events accompanying neuronal activation (Sheng and Greenberg 1990). Thus c-Fos expression can serve as a starting point for the analysis of intracellular events within phenotypically-distinct neurons activated during CTA expression.

4.The patterns of c-Fos expression can be interpreted against a large database of c-Fos literature. For example, c-Fos has been used extensively to map the sites involved in CTA acquisition and expression [56-62], and the functional connections of the vestibular system [2, 48-51, 63-71]. Thus the c-Fos patterns (induced by MF exposure that also induces CTA and causes vestibular disturbances) will be immediately interpretable.

5. Finally, c-Fos is itself a transcription factor that regulates the expression of target genes(Morgan and Curran 1991)involved in normal growth and plasticity during development (Grigoriadis, Wang et al. 1994), learning(Kaang, Kandel et al. 1993) , and recovery from injury (Jones and Evinger 1991; Weiser, Baker et al. 1993). It is a goal of our research to determine if c-Fos plays a physiological role in CTA by regulating gene expression and protein synthesis in specific brain regions critical for CTA acquisition or expression.

As a marker of neural activity after magnetic field stimulation, c-Fos offers another advantage:

6. c-Fos is a delayed marker of brain activation. The same synaptic activity and intracellular cascades that mediate the acute processes of neuronal activity and behavior at the time of stimulation may also initiate the slower processes of immediate-early gene activation and protein synthesis. Activation of the c-Fos gene by a sensory stimulus results in c-Fos protein synthesis within 1h [54]. Because c-Fos is visualized 1 h after stimulation, there will be no interference of the magnet apparatus or MF stimulus with the visualization procedure.

Disadvantages of c-Fos

c-Fos expression also has disadvantages. It is a postmortem technique, so the same rat cannot be repeatedly tested. Also, only a subset of activated neurons may be visualized, because some cells do not express c-Fos when activated. Although there are other ways to record or visualize neural activity that avoid these problems, most are impractical when applied to high MFs. Electrophysiological recordings using surface or indwelling metallic electrodes are confounded either by magnetic attraction or by induced currents. Functional MRI, in addition to having relatively low-resolution in small animals, is obviously confounded by the presence of the MF if the field itself is inducing neuronal activity. Other methods of measuring brain activity, such as brain imaging by PET or voltage-sensitive dyes, or neurotransmitter release by microdialysis, would be prohibitively cumbersome to conduct within the confines of the magnet’s bore.

References

Brozek, G., O. Buresova, et al. (1974). "Effect of bilateral cortical spreading depression on the hippocampal theta activity induced by oral infusion of aversive gustatory stimulus." Expt. Neurol. 42: 661-668.

Buresova, O., Z. A. Aleksanyan, et al. (1979). "Electrophysiological analysis of retrieval of conditioned taste aversion in rats. Unit activity changes in critical brain regions." Physiol. Bohemoslov. 28: 525-536.

Chang, F.-C. T. and T. R. Scott (1984). "Conditioned taste aversions modify neural responses in the rat nucleus tractus solitarius." J. Neurosci. 4: 1850-1862.

Cirelli, C., M. Popmeiano, et al. (1996). "c-Fos expression in the rat brain after unilateral labyrinthectomy and its relation to the uncompensated and compensated stages." Neurosci. 70: 513-546.

Darlington, C. L., P. Lawlor, et al. (1996). "Temporal relationship between the expression of Fos, Jun and Krox-24 in the guinea pig vestibular nuclei during the development of vestibular compensation for unilateral vestibular deafferentation." Brain Res 735: 173-176.

DiLorenzo, P. M. (1985). "Responses to NaCl of parabrachial units that were conditioned with intravenous LiCl." Chem. Senses 10: 438.

Garcia, J. and R. A. Koelling (1966). "Relation of cue to consequence in avoidance learning." Psychonomic Sci. 4: 123-124.

Gould, J. L. (1998). "Sensory bases of navigation." Current Biology 8: R731-738.

Grigoriadis, A. E., Z.-Q. Wang, et al. (1994). "c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling." Sciecne 266: 443-448.

Gustave Dit Duflo, S., C. Gestreau, et al. (1999). "Fos expression the cat brainstem after unilateral vestibular neurectomy." Brain Res 824: 1-17.

Houpt, T. A., R. A. Berlin, et al. (1997). "Subdiaphragmatic vagotomy does not attenuate c-Fos induction in the nucelus of the solitary tract after conditioned taste aversion expression." Brain Res. 747: 85-91.

Houpt, T. A., J. M. Philopena, et al. (1995). "c-Fos expression in the nucleus of the solitary tract following conditioned taste aversion formation." Appetite 24: 84.

Houpt, T. A., J. M. Philopena, et al. (1996). "c-Fos induction in the rat nucleus of the solitary tract by intraoral quinine infusion depends on prior pairing of quinine and lithium chloride." Physiol. Behav. 60: 1535-41.

Houpt, T. A., J. M. Philopena, et al. (1996). "c-Fos induction in the rat nucleus of the solitary tract correlates with the retention and forgetting of a conditioned taste aversion." Learn. Mem. 3: 25-30.

Houpt, T. A., J. M. Philopena, et al. (1994). "Increased c-Fos expression in the rat nucleus of the solitary tract after conditioned taste aversion formation." Neurosci. Lett. 172: 1-5.

Jones, K. J. and C. Evinger (1991). "Differential neuronal expression of c-fos proto-oncogene following peripheral nerve injury or chemically-induced seizure." J. Neurosci.Res. 28: 291-298.

Kaang, B.-K., E. R. Kandel, et al. (1993). "Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons." Neuron 10: 427-435.

Kangarlu, A., r. E. Burgess, et al. (1999). "Cognitive, cardiac, and physiological safety studies in ultra high field magnetic resonance imaging." Magn. Reson. Imag. 17: 1407-1416.

Kaufman, G. D. (1996). "Activation of immediate-early genes by vestibular stimulation." Ann. NY Acad. Sci. 781: 437-442.

Kaufman, G. D., J. H. Anderson, et al. (1991). "Activation of a specific vestibulo-olivary pathway by centripetal acceleration in rat." Brain Res. 562: 311-317.

Kaufman, G. D., J. H. Anderson, et al. (1992). "Fos-defined activity in rat brainstem following centripetal acceleration." J. Neurosci. 12: 4489-4500.

Kaufman, G. D., J. H. Anderson, et al. (1993). "Otolith-brain stem connectivity: evidence for differential neural activation by vestibular hair cells based on quantification of Fos expression in unilateral labyrinthectomized rats." J. Neurophysiol. 70: 117-127.

Kaufman, G. D. and A. A. Perachio (1994). "Translabyrinth electrical stimulation for the induction of immediate early genes in the gerbil brainstem." Brain Res. 646: 345-350.

Kim, M. S., B. K. Jin, et al. (1997). "Effect of MK801 on cFos-like protein expression in the medial vestibular nucleus at early stage of vestibular compensation in uvulonodullectomized rats." Neurosci. Lttrs. 231: 147-150.

Kitahara, T., T. Saika, et al. (1995). "Changes in fos and jun expression in the rat brainstem in the process of vestibular compensation." Acta Otolaryngol. 520: S401-S404.

Kitahara, T., N. Takeda, et al. (1995). "Effects of MK801 on Fos expression in the rat brainstem after unilateral labyrintectomy." Brain Res. 700: 182-190.

Kitahara, T., N. Takeda, et al. (1997). "Role of the flocculus in the development of vestibular compensation: immunohistochemical studies with retograde tracing and flocculectomy using fos expression as a marker in the rat brainstem." Neurosci. 76: 571-580.

Mark, G. P., P. Rada, et al. (1992). "Cholinergic activity in the nucleus accumbens may be inversely related to food intake." Appetite 19: 201.

Marshburn, T. H., G. D. Kaufman, et al. (1997). "Saccule contribution to immediate early gene induction in the gerbil brainstem with posterior canal galvanic or hypergravity stimulation." Brain Res. 761: 51-58.

Morgan, J. I. and T. Curran (1991). "Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun." Ann. Rev. Neurosci. 14: 421-451.

Nolte, C. M., D. W. Pittman, et al. (1998). "Magnetic field conditioned taste aversion in rats." Physiol. Behav. 63: 683-688.

Rosenblum, K., D. E. Berman, et al. (1997). "NMDA receptor and the tyrosine phosphorylation of its 2B subunit in taste learning in the rat insular cortex." J. Neurosci. 17: 5129-5135.

Sagar, S. M., F. R. Sharp, et al. (1988). "Expression of c-fos protein in brain: metabolic mapping at the cellular level." Science 240: 1328-1331.

Sato, T., W. Tokuyama, et al. (1997). "Temporal and spatial dissociation of expression patterns between Zif268 and c-Fos in rat inferior olive during vestibular compensation." NeuroReport 8: 1891-1895.

Schafe, G. E. and I. L. Bernstein (1996). "Forebrain contribution to the induction of a brainstem correlate of conditioned taste aversion: I. The amygdala." Brain Res. 741: 109-116.

Schafe, G. E. and B. I.L. (1998). "Forebrain contribution to the induction of a brainstem correlate of conditioned taste aversion. II. Insular (gustatory) cortex." Brain Res. 800: 40-47.

Schafe, G. E., R. J. Seeley, et al. (1995). "Forebrain contributions to the induction of a cellular correlate of conditioned taste aversion in the nucleus of the solitary tract." J. Neurosci. 15: 6789-6796.

Schenck, J. F., C. L. Dumoulin, et al. (1992). "Human exposure to 4.0-Tesla magnetic fields in a whole body scanner." Med. Phys. 19: 1089-1098.

Sheng, M. and M. E. Greenberg (1990). "The regulation and function of c-fos and other immediate early genes in the nervous system." Neuron 7: 477-485.

Swank, M. W. and I. L. Bernstein (1994). "c-Fos induction in response to a conditioned stimulus after single trial taste aversion learning." Brain Res. 636: 202-208.

Swank, M. W., A. E. Ellis, et al. (1996). "c-Fos antisense blocks acquisition and extinction of conditioned taste aversion in mice." Neuroreport 7: 1866-1870.

Swank, M. W., G. E. Schafe, et al. (1995). "c-Fos induction in response to taste stimuli previously paired with amphetamine or LiCl during taste aversion learning." Brain Res. 673: 251-261.

Thiele, T. E., M. F. Roitman, et al. (1996). "c-Fos induction in rat brainstem in response to ethanol- and lithium chloride-induced conditioned taste aversions." Alcohol. Clin. Exp. Res. 20: 1023-1028.

Walker, M. M., C. E. Diebel, et al. (1997). "Structure and function of the vertebrate magnetic sense." Nature 390: 371-376.

Weiser, M., H. Baker, et al. (1993). "Axotomy-induced differential gene induction in neurons of the locus ceruleus and substantia nigra." Mol. Brain Res. 17: 319-327.

Yamamoto (1989). "Taste responses of cortical neurons in freely ingesting rats." J. Neurophysiol. 61: 1244-1258.

Yamamoto, T., T. Shimura, et al. (1991). "Role of the amygdala in conditioned taste avesion learning in the rat." Neurosci. Res. Suppl. 16: S140.