Justification of C-Fos as a Marker of Neuronal Activity

C Fos

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Justification of c-Fos as a Marker of Neuronal Activity

From Magnet Grant:

Analysis of Magnetic Field Detection. Four criteria must be met to demonstrate that an animal can detect and respond to MFs: 1. the MF must cause a behavioral response. 2. neuronal activity must be correlated with the presence of the MF. 3. the receptive sensory organ must be identified. 4. the intracellular transduction mechanism of the field must be determined. Although there are many examples of lower vertebrates responding to MFs, e.g. for navigation during migration (Gould 1998), these four criteria have not been met for any vertebrate. The model that has come the closest to meeting all 4 criteria is in the trout (Walker, Diebel et al. 1997): 1. the fish can be operantly conditioned to associate a 50 µT MF with food reward, 2. MF-induced activity in the trigeminal nerves has been electrophysiologically recorded, and 3.tracing studies have demonstrated that the trigeminal nerves project to the basil lamina of the olfactory epithelium, the putative receptive organ, and 4. crystals of magnetite have been visualized within cells of the olfactory epithelium, providing a potential substrate for intracellular transduction of the MF.

We will apply the same set of criteria to the analysis of static MF effects on rats. Our marker of a behavioral response will be both direct observation of post-exposure behaviors, and the expression of CTA. Our marker of neuronal activity will be c-Fos expression in the brain. We will use mutations of the inner ear to target potential receptive organs, and pharmacology to identify neurochemical substrates of MF transduction. The identification of the receptive organ will suggest possible cellular and intracellular transduction mechanisms, although intracellular analysis lies beyond the scope of this proposal.

Conditioned Taste Aversion as a Behavioral Marker. As a form of associative learning by which an animal avoids a novel taste or food that has been previously paired with a toxin, CTA has been widely used as a marker of aversive effects caused by drugs and treatments. CTA learning is remarkably sensitive, and often reveals a treatment effect even when no other behavioral effect is detectable. Purely exteroceptive sensory stimuli, such as light or auditory cues produced by the MF or the apparatus, are likely to be ignored because CTAs overwhelmingly favor a novel taste as the conditioned stimulus (Garcia and Koelling 1966). Likewise, stressful effects of the exposure procedure, such as restraint or cutaneous discomfort, are not sufficient to act as the unconditioned stimulus in CTA learning (Garcia and Koelling 1966; Nolte, Pittman et al. 1998). Aversive effects that activate interoreceptors or induce nausea in humans, however, are favored to induce CTAs (Garcia and Koelling 1966); this increases the likelihood of measuring a behavioral effect that can be correlated with human self-reports of vertigo and nausea in high MFs (Schenck, Dumoulin et al. 1992; Kangarlu, Burgess et al. 1999).

c-Fos Expression as a Neural Marker. As a marker of neural activity, c-Fos offers several advantages:

1. c-Fos is a delayed marker of brain activation. The same neuronal process that mediate the rapid 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 (Morgan and Curran 1991). 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.

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

3.c-Fos allows mapping of neuronal populations activated by a stimulus. The central processing of gustatory, visceral and vestibular sensation is mediated by many nuclei throughout the brain. Because many sections can be processed for c-Fos, activity in multiple brain regions of the same animal can be visualized for the analysis of a distributed network.

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 (Houpt, Philopena et al. 1994; Swank and Bernstein 1994; Houpt, Philopena et al. 1995; Swank, Schafe et al. 1995; Houpt, Philopena et al. 1996; Houpt, Philopena et al. 1996; Swank, Ellis et al. 1996), and the functional connections of the vestibular system (Kaufman, Anderson et al. 1991; Kaufman, Anderson et al. 1992; Kaufman, Anderson et al. 1993; Kaufman and Perachio 1994; Kitahara, Saika et al. 1995; Kitahara, Takeda et al. 1995; Cirelli, Popmeiano et al. 1996; Darlington, Lawlor et al. 1996; Kaufman 1996; Kim, Jin et al. 1997; Kitahara, Takeda et al. 1997; Marshburn, Kaufman et al. 1997; Sato, Tokuyama et al. 1997; Gustave Dit Duflo, Gestreau et al. 1999). Thus the c-Fos patterns (induced by MF exposure that also induces CTA and causes vestibular disturbances) will be immediately interpretable.

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.

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From CTA Grant:

Summary of Lesion Studies. The literature on the effects of lesions on CTA are consistent with distributed processing of gustatory and of toxic stimuli, with different brain loci responsible for signal transduction,