Immunocytochemical detection for the up-regulation of c-fos is a popular method to reveal the physiological identity of neurones in fixed tissue. We have developed a novel genetic tool that allows tracking of c-fos expression in living cells. The construct comprises 1095 bp of the promoter sequence from the mouse c-fos gene driving expression of EGFP. This construct was engineered into a replication-deficient adenoviral vector (AdmfosEGFP) and tested in experiments in vitro and in vivo. All experiments conformed to Home Office guidelines.
For in vivo experiments, seven male Wistar rats (250-275 g) were anaesthetised (medetomidine HCl, 0.6 mg kg-1 I.P. and ketamine HCl, 100 mg kg-1 I.P.) and AdmfosEGFP was introduced by stereotaxic injection into the paraventricular nucleus of the hypothalamus (PVN). Magnocellular neurones in this region respond to dehydration and low blood volume. Rats were allowed to recover for 2 weeks before an osmotic challenge with 1.5 M NaCl I.P. (following the protocol of Wang et al. 1997). Following a period of 5 h, rats were terminally anaesthetised with pentobarbitone, transcardially perfused, fixed and the brain was removed. Fluorescence photomicrographs were taken from 40 µm sections through the rostrocaudal extent of PVN, from both osmotically challenged (n = 3) and control animals (n = 4). The average intensity of green pixels relative to black background in the area of PVN was calculated using Corel Photo-Paint. The osmotic challenge significantly increased the amount of EGFP-related green fluorescence (Mann-Whitney test, P < 0.05).
For in vitro application, organotypic slice cultures were prepared from the hypothalamic region of P8 Wistar rats, which were halothane-anaesthetised and decapitated. Slices specifically included the PVN, and were transfected with AdmfosEGFP. The slices were plated on a Millicell membrane (Millipore) and left for 2 weeks. A single drop of 30 mM KCl was used as stimulus to trigger Ca2+ release unilaterally. Three hours later, fluorescent cells in the region of the PVN were visualised under a confocal microscope. Stimulation caused a dramatic increase in numbers of fluorescent cells on the stimulated side (386 ± 279 % (mean ± S.E.M.), P < 0.001, n = 7; Student’s paired, 2-tailed, t test).
In conclusion, AdmfosEGFP allows detection of c-fos activation by monitoring of EGFP fluorescence, and opens up the possibility to study the dynamics of c-fos expression in living tissue for the first time.
C.E.L.S. is funded by a University of Bristol PhD Studentship. The BHF and Royal Society funded research.