(A) Expression of c-fos mRNA in adult rat brain after ECS and rTMS. Results of in situ hybridization analyses using an α-[³⁵S]thio-dATP-labeled oligonucleotide probe are shown. Coronal sections of control rats (Top) and rats subjected to ECS (Middle) or rTMS (Bottom) are depicted. ECS and rTMS evoke different patterns of c-fos expression: ECS produces a strong increase of c-fos mRNA expression throughout the cortex and hippocampus, whereas the highest response to rTMS can be observed in thalamic regions, as indicated by an arrow (Bottom). (Scale bar = 2 mm.) (B) Comparative analyses of c-fos mRNA expression after ECS and rTMS in cerebral cortex, cingulate cortex, and hippocampus, as detected by nonradioactive in situ hybridization. ECS induces strong c-fos mRNA expression in all these regions. In contrast, rTMS evokes c-fos expression predominantly in the medial (arrowheads) and cingulate (arrow) cortex. No detectable increase of c-fos expression was observable in hippocampus after rTMS. (Scale bars = 1 mm in all rows.)

Immunocytochemical staining of the PVT with c-Fos- and c-Jun-specific antibodies after ECS, rTMS, and either one or three successive applications of rTMS. Background levels of c-Fos-positive nuclei in the PVT are very low (Control, Top). rTMS produces a robust c-Fos induction in PVT neurons (one time rTMS, Top). Three trains of rTMS result in a much higher induction of c-Fos-positive nuclei in this region than a single application (three times rTMS, Middle). ECS produces only a small increase in c-Fos expression in PVT neurons (one time ECS, Middle). The induction of another immediate early gene (c-Jun) was also analyzed: as for c-Fos, rTMS increases c-Jun expression in PVT neurons (one time rTMS, Bottom). However, the background levels of c-Jun expression in these neurons are higher than those of c-Fos (Control, Bottom). (Scale bar = 0.5 mm.)

Patterns of c-fos expression induced by rTMS from different orientations in vivo and in brain slices. (A) rTMS elicits the same patterns of c-fos expression, as shown with nonradioactive in situ hybridization, when the stimulating coil is applied to the rat head dorsally (Upper Left) or ventrally (Upper Right). (Scale bar = 0.5 mm.) (B) rTMS is able to stimulate PVT neurons in vitro. Results of a representative experiment are shown. Strong c-fos expression is detected in PVT neurons with nonradioactive in situ hybridization in stimulated brain slices kept in an interface chamber. Experiments were repeated three times. (Scale bar = 0.5 mm.) (C) Extracellular field potentials recorded from coronal brain slices, to test for viability. An extracellular glass recording pipette was placed either in area CA1 of the hippocampus (trace 1) or in the superficial layers of the neocortex (trace 2). Stimulating electrodes were placed in either the hippocampal Schaffer collaterals or in the subcortical white matter. The resulting extracellular field potentials are shown (average of 2). The recordings were performed before application of magnetic stimulation, but at least 3 hr after the slices were made in a standard interface chamber at 33°C.

rTMS activates brain regions controlling circadian rhythms. Induction of c-fos mRNA expression after rTMS can be detected not only in the paraventricular nucleus of the thalamus (PVT) but also in the suprachiasmatic nucleus (SCN) and the paraventricular nucleus of the hypothalamus (PVN). Background levels of c-fos mRNA expression in corresponding unstimulated regions are depicted on the left. (Scale bars = 0.5 mm in Top and 0.1 mm in Middle and Bottom.)

Comparative analysis of the effects of rTMS and light exposure on pinealocytes and retina. (A) Effects of rTMS on pineal gland and retina during the light phase of the circadian rhythm. Western blot analysis of pineal gland and retinal extracts with antisera specific for the phosphorylated form of CREB (pCREB) is shown. Background levels of pCREB in pineal and retina are shown in lanes 1. rTMS at 10% power does not increase pCREB immunoreactivity (lanes 2), whereas rTMS at 100% power is able to induce strong CREB phosphorylation within 15 min (lanes 3). At 45 min pCREB immunoreactivity is reduced in both tissues (lanes 4). Experiments were performed three times. (B) rTMS and light stimulation in the dark phase of the circadian rhythm produce similar changes in c-fos expression in pinealocytes and PVT neurons. Stimulations were performed at 2:00 a.m. (rTMS as described in Materials and Methods; light stimulation 10 min, 40 W). (Top) c-fos mRNA expression in control regions. (Middle and Bottom) c-fos expression in the pineal gland and in the PVT after rTMS and light stimulation, respectively. Both stimulation modalities produce a strong reduction of c-fos expression in pinealocytes and a robust induction of c-fos expression in PVT neurons.