Abstract
Fos-Tau-LacZ (FTL) transgenic mice are used to visualize the anatomical connectivity of neurons that express c-Fos, an immediate early gene, in response to activation. In contrast to typical c-Fos protein expression, which is localized to the nucleus of stimulated neurons, activation of the c-Fos gene results in beta galactosidase (β-gal) expression throughout the entire cytoplasm of activated cells in FTL mice; thereby making it possible to discern the morphology of c-Fos expressing cells. This can be an especially important tool in brain areas in which function may be related to cell morphology, such as the primary taste/viscerosensory brainstem nucleus of the solitary tract (nTS). Thus, to further characterize FTL activity in the brain, the current study quantified both β-gal enzymatic activity as well as c-Fos protein expression in the nTS under a variety of experimental conditions (no stimulation, no stimulation with prior overnight food and water restriction, monosodium glutamate taste stimulation, and monosodium glutamate taste stimulation with perfusion 5 h post stimulation). Contrary to previous research, we found that β-gal activity (both labeled cell bodies and overall number of labeled pixels) was unchanged across all experimental conditions. However, traditional c-Fos protein activity (both cell bodies and number of activated pixels) varied significantly across experimental conditions, with the greatest amount of c-Fos protein label found in the group that received monosodium glutamate taste stimulation. Interestingly, although many c-Fos positive cells were also β-gal positive in the taste stimulated group, some c-Fos protein labeled cells were not co-labeled with β-gal. Together, these data suggest that β-gal staining within the nTS reflects a stable population of β-gal- positive neurons whose pattern of expression is unaffected by experimental condition.
Highlights
The immediate early gene, c-Fos, has been used extensively as an anatomical marker of neuronal activity since the early 1980s [1], with over 19,000 publications employing this technique
This is of particular importance in brain areas whose function may be dependent upon both regional localization and cell morphology, such as the nucleus of the solitary tract
Traditional c-Fos expression is confined to the nucleus of activated cells, making it impossible to discern the morphology of activated cells
Summary
The immediate early gene, c-Fos, has been used extensively as an anatomical marker of neuronal activity since the early 1980s [1], with over 19,000 publications employing this technique. Use of c-Fos as a neuronal activity marker enables visualization of spatial patterns of brain activation throughout a region of interest as well as the co-localization of secondary and tertiary cellular markers that c-Fos positive cells express (e.g. neuropeptides, enzymes, etc.,), making identification of an activated cell’s neuronal phenotype possible. Because c-Fos protein staining is limited to the nucleus of activated cells, determination of the morphology of activated cells remains unknown. This is of particular importance in brain areas whose function may be dependent upon both regional localization and cell morphology, such as the nucleus of the solitary tract (nTS; primary taste/viscerosensory nucleus; [2]). Because the b-gal enzyme is first synthesized prior to transportation throughout the cytoplasm of activated cells, this results in a delay in b-gal activity compared to c-Fos protein (b-gal expression peaks +4 hours post stimulation, whereas c-Fos protein expression peaks 1–3 hours following stimulation; [4,5])
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