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
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.
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, sensory processing, integration and association, and final output. Lesions of the input pathways responsible for transducing the toxic unconditioned stimuli block acquisition; lesions of the taste input pathways either attenuate responsiveness to tastants or block CTA acquisition (e.g. mPBN). Lesions of more rostral projection sites in the gustatory cortex and amygdala attenuate acquisition or expression of CTAs, possibly by injuring the mechanisms of association, consolidation and retention. Thus, the medial PBN appears to be a necessary relay during acquisition, while the gustatory cortex emerges as an apparently necessary site for mediating acquisition and expression of CTA. The amygdala also appears to be necessary for full acquisition and expression, but the precise role of specific amygdalar subnuclei and connections in CTA learning is still unclear.
What is not clear from previous studies, however, is whether lesions that produce a deficit in CTA learning indicate localization of function in the site of injury or whether the lesions reveal necessary connections passing through or relayed by the lesion site from distant sites. Even if the cells in a brain site prove necessary, it is still possible that the site is a relay between other sites, and that no associative plasticity occurs there during CTA learning. Fixing the role of a site within the network requires tracking correlates of activity through the network.
Neuronal Correlates of CTA Expression. There are only a few scattered reports, however,
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.
c-Fos Expression as a Correlate of CTA Expression. 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 as a Novel Approach to the Neurobiology of 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. 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.
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