Neural circuit repair by low-intensity magnetic stimulation requires cellular magnetoreceptors and specific stimulation patterns

Abstract

Although electromagnetic brain stimulation is a promising treatment in neurology and psychiatry, clinical outcomes are variable, and underlying mechanisms are ill-defined, which impedes the development of new effective stimulation protocols. Here, we show, in vivo and ex vivo, that repetitive transcranial magnetic stimulation at low-intensity (LI-rTMS) induces axon outgrowth and synaptogenesis to repair a neural circuit. This repair depends on stimulation pattern, with complex biomimetic patterns being particularly effective, and the presence of cryptochrome, a putative magnetoreceptor. Only repair-promoting LI-rTMS patterns up-regulated genes involved in neuronal repair; almost 40% of were cryptochrome targets. Our data open a new framework to understand the mechanisms underlying structural neuroplasticity induced by electromagnetic stimulation. Rather than neuronal activation by induced electric currents, we propose that weak magnetic fields act through cryptochrome to activate cellular signaling cascades. This information opens new routes to optimize electromagnetic stimulation and develop effective treatments for different neurological diseases.

Fig. 1. LI-rTMS induces transcommissural climbing fiber reinnervation in pedunculotomized adult mice.

(A) Climbing fibers (white arrows) in the molecular layer (ML) of the intact right hemisphere (CRUS 1 and CRUS 2 coronal sections) from a sham-treated mouse (n = 5). (B) After sham treatment, in coronal sections, VGLUT2-positive climbing fibers (white arrows) in the molecular layer of the right hemicerebellum stop at the midline (thick vertical dashed line), consistent with lack of reinnervation (18). (C) In comparison after LI-rTMS (n = 9), VGLUT2-positive climbing fibers (white arrows) fill the molecular layer on both sides of the midline (thick vertical dashed line) in this coronal section, consistent with reinnervation. (D) Further laterally, VGLUT2-positive climbing fibers (white arrows) in the molecular layer of the lesioned hemisphere following LI-rTMS. Anatomical differences from (A) and (B) reflect the noncoronal orientation of the lobule (simplex), and therefore, climbing fiber arbors are angled to the coronal plane of the section. (E) Diagram showing the coil (blue) in relation to the mouse head. (F) Unfolded cerebellum showing magnetic field intensity delivered by LI-rTMS, as measured by a Hall device in air at corresponding X, Y, and Z distances from the center of the coil. (G) Average density, in 0.5-mm parasagittal zones, of LI-rTMS–induced climbing fiber reinnervation (n = 9). This parasagittal organization of different reinnervation densities is consistent with that previously demonstrated in BDNF-induced climbing fiber reinnervation, which demonstrates parasagittal topography and provides recovery of motor and navigation behaviors (18). Grayscale grading: 1 = few strands; 2 = one-quarter lobule; 3 = half lobule; 4 = three-quarter lobule; and 5 = completely climbing fiber–filled (18). GL, granular layer; LHCbm, left hemicerebellum; RHCbm, right hemicerebellum; SIM, lobulus simplex; PM, paramedian lobule; CoP, copula pyramidis; I to X, lobules 1 to 10 of the vermis. Photo credit: A. D. Tang, University of Western Australia, Experimental and Regenerative Neuroscience Laboratory.

Fig. 2. LI-rMS pattern regulates climbing fiber reinnervation ex vivo.

(A) Hemicerebella are removed from an explant (dotted line) and placed next to an intact explant (dashed arrows) for reinnervation (red dotted arrows) by host climbing fibers (thin gray arrows). , LI-rMS (see fig. S1). (B) Purkinje cell (green) showing climbing fiber reinnervation (red puncta, arrows). Photo credit: R. M. Sherrard, Sorbonne Université, UMR8256 Biological Adaptation and Ageing. (C) Purkinje cell (PC) reinnervation is greater in proximal versus distal zones of the cerebellar plate [two-way repeated-measures analysis of variance (ANOVA), P < 0.001]. BHFS (B; n = 11) and intermittent theta burst stimulation (iTBS) (iT; n = 8) induced significant reinnervation in both zones compared with sham (S; n = 10; ANOVA with Tukey post hoc; proximal: BHFS and iTBS, both P < 0.001; distal: BHFS, P = 0.003; iTBS, P = 0.002). Ten hertz (n = 8) also induced Purkinje cell reinnervation proximally (P = 0.048), but not distally (P = 0.96), although less than iTBS and BHFS (P < 0.001). Excitatory and inhibitory indicate stimulus effects in high-intensity rTMS [see (E)]. One Hz (1; n = 6), continuous theta burst stimulation (cTBS) (cT; n = 8), and randomized iTBS (R-iTBS) (R-iT; n = 7) did not induce more reinnervation than sham (proximal: 1 Hz, P = 0.577; cTBS, P = 0.097; R-iTBS, P = 0.952; distal: 1 Hz, P = 0.98; cTBS, P = 0.95; R-iTBS, P = 0.93). Bars = means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Reinnervation density does not reflect the number of pulses delivered per 10-min session (Pearson coefficient, P = 0.353), although changes in patterns may also contribute to this effect. (E) Pulses delivered in 10 min for each stimulation parameter and their effects in high-intensity rTMS. ■, Sham; □, 1 Hz; , 10 Hz; , BHFS; , iTBS; , cTBS; , R-iTBS.

Fig. 3. LI-rMS intensity has a lower limit below which reinnervation fails.

(A) BHFS stimulation of either the rostral [cerebellum (Cbm); n = 4] or the caudal (ION; n = 4) part of the explant does not induce significant reinnervation compared with sham (ANOVA with Tukey post hoc; cerebellum: P = 0.20; ION: P = 0.12). “All” is for comparison, showing reinnervation induced by BHFS (***P < 0.001) to the whole explant (Fig. 2). (B) Distribution of the magnetic (|B|; top) and electric (|E|; bottom) fields in the explant without shielding [reproduced from (20) with permission]. (C) Distribution of the modeled electric field (|E|) and magnetic field (|B|) in the explant when the caudal part is shielded (“cerebellum”; left) or when the rostral part is shielded (“ION”; right) from the LI-rMS coil. Arrowheads indicate the direction of the induced current flow or magnetic flux, as appropriate; and solid white lines delineate the areas of interest for stimulation (cerebellar plates or inferior olives).

Fig. 4. LI-rMS modulates gene expression in denervated hemicerebellar plates.

(A) c-fos (red) in Purkinje cell (cyan) and GABAergic (GABA-releasing) interneuron (green) nuclei (arrows). Scale bars, 20 μm. Photo credit: T. Dufor, Sorbonne Université, UMR 8256 Biological Adaptation and Ageing. PV, parvalbumin. (B) c-fos–positive profiles per square millimeter (means ± SEM) in sham (n = 8), BHFS (n = 6), iTBS (n = 9), R-iTBS (n = 4), or cTBS (n = 7) hemicerebella (ANOVA and Tukey post hoc comparison, F_4,29 = 7.3, P < 0.001; compared to sham: BHFS, P = 0.001; iTBS, P = 0.16; R-iTBS, P < 0.001; cTBS, P = 0.68). (C) c-fos labeling does not reflect stimulation pulse number (Pearson coefficient, P = 0.30), although changes in patterns may also contribute to this effect; color codes are the same as in (B). (D) Percentage of calbindin- or parvalbumin-positive cells that are double-labeled c-fos/calbindin (“PCs”) or c-fos/parvalbumin (“INs”; means ± SEM) in sham controls or after BHFS, iTBS, or R-iTBS (Fisher’s exact test, compared with sham; BHFS: Purkinje cells, P < 0.001; interneurons, P = 0.006; iTBS: Purkinje cells, P < 0.001; interneurons, P = 0.001; R-iTBS: Purkinje cells, P = 0.827; interneurons, P = 0.682; cTBS: Purkinje cells, P = 0.50; interneurons, P = 0.61). (E) Heatmap showing expression changes of 22 genes from the mouse neurotrophins and receptors polymerase chain reaction (PCR) array after BHFS, iTBS, or R-iTBS. Red, up-regulated (P < 0.05); orange, strong trend for up-regulation (0.05 < P < 0.1); royal blue, down-regulated (P < 0.05); light blue, strong tend for down-regulation (0.05 < P < 0.1); gray, no change. CB, calbindin. Compared with sham, **P < 0.01, ***P < 0.001. Color codes are the same as in (B).

Fig. 5. Cryptochromes are required for LI-rMS–induced postlesion repair.

Percentage of reinnervated PCs in the proximal zone of grafted cerebellar plates from explants of WT or Cry1^−/−Cry2^−/− (DKO) embryos. In DKO explants, BHFS (n = 7) did not induce reinnervation compared with sham (n = 10) (one-way ANOVA with Tukey post hoc; P = 0.946), whereas BDNF (n = 7) did (P < 0.001). “WT BHFS” is given for comparison from Fig. 2.