Remotely controlled chemomagnetic modulation of targeted neural circuits

Abstract

Connecting neural circuit output to behaviour can be facilitated by the precise chemical manipulation of specific cell populations^1,2. Engineered receptors exclusively activated by designer small molecules enable manipulation of specific neural pathways^3,4. However, their application to studies of behaviour has thus far been hampered by a trade-off between the low temporal resolution of systemic injection versus the invasiveness of implanted cannulae or infusion pumps². Here, we developed a remotely controlled chemomagnetic modulation-a nanomaterials-based technique that permits the pharmacological interrogation of targeted neural populations in freely moving subjects. The heat dissipated by magnetic nanoparticles (MNPs) in the presence of alternating magnetic fields (AMFs) triggers small-molecule release from thermally sensitive lipid vesicles with a 20 s latency. Coupled with the chemogenetic activation of engineered receptors, this technique permits the control of specific neurons with temporal and spatial precision. The delivery of chemomagnetic particles to the ventral tegmental area (VTA) allows the remote modulation of motivated behaviour in mice. Furthermore, this chemomagnetic approach activates endogenous circuits by enabling the regulated release of receptor ligands. Applied to an endogenous dopamine receptor D1 (DRD1) agonist in the nucleus accumbens (NAc), a brain area involved in mediating social interactions, chemomagnetic modulation increases sociability in mice. By offering a temporally precise control of specified ligand-receptor interactions in neurons, this approach may facilitate molecular neuroscience studies in behaving organisms.

Figure 1. Magnetically controlled chemical payload release.

a, Experimental scheme of alternating magnetic field (AMF)-triggered chemical payload release from the magnetoliposomes. b, Transmission electron microscope (TEM) images and the size distributions from dynamic light scattering spectra of the magnetic nanoparticles (MNPs) before (I) and after (II) encapsulation into the magnetoliposomes, and the magnetoliposomes after exposure to 40 s of AMF (H₀=45±2 mT and ƒ=150 kHz) (III). Scale bar: 200nm. c, Fluorescent dye (Alexa Fluor® 488) release from magnetoliposomes (mean ± standard deviation, s.d., n=3 independent samples) during the bath temperature increase from 37°C to 43°C. d, AMF-triggered dye release (mean ± s.d., n=3 independent samples) from magnetoliposomes and the temperature profile of the bulk solution (solid line: mean, shaded area: s.d. n=3). e, Confocal images of the primary hippocampal neurons expressing hSyn::hM3D(Gq)-mCherry and hSyn-GCaMP6s. Scale bar: 50μm. The experiment was repeated three times independently with similar results. f-i, Heat maps of normalized GCaMP6s fluorescence intensity of 100 automatically selected neurons in different experimental conditions. F₀ is defined as the mean of the fluorescence intensity during the first 10 s. ON: AMF is turned on. OFF: AMF is turned off. AMF conditions: H₀=45±2 mT, ƒ=150 kHz, 20 s.

Figure 2. Chemomagnetic stimulation in vivo.

a, Experimental timeline for the viral gene delivery, magnetoliposome injection, and AMF stimulation. Inset: A confocal image of the expression of hM3D(Gq)-mCherry in the mouse VTA. Scale bar: 200 μm. b-d, Left: Confocal images of the c-fos expression in the VTA (b), NAc (c) and mPFC (d) of mice exposed to AMF (AMF⁺), injected with CNO-loaded magnetoliposomes (CNO⁺) and expressing hM3D(Gq) (hM3D(Gq)⁺). Scale bar: 50 μm. Right: the percentages of c-fos expressing (c-fos⁺) neurons among DAPI-labelled cells in each group (mean ± standard error of the mean, s.e.m.). Increased c-fos expression is observed following chemomagnetic treatment as confirmed by one-way ANOVA and Turkey’s multiple comparisons test (n=5 mice, VTA F_{3, 16} = 86.29, NAc F_{3, 16} = 207.6, mPFC F_{3, 16} =30.97, **** p<0.0001). All c-fos quantification experiments were conducted in anesthetized mice. e, Photometry setup integrated with an AMF coil. f, Confocal images of co-expression of GCaMP6s and hM3D(Gq)-mcherry in the mouse VTA. The experiment was repeated three times independently with similar results. Scale bar: 50 μm. g, Normalized dynamic fluorescence intensity change (ΔF/F₀) of GCaMP6s in the VTA of mice freely moving within the AMF coil. Fluorescence increase was observed only upon applying AMF stimulation in mice expressing hM3D(Gq) and injected with CNO-loaded magnetoliposomes (red). The blue area represents AMF exposure. In all experiments H₀ =45±2 mT, ƒ = 164 kHz. Solid lines: mean, shaded areas: s.e.m., n=3 mice for each test condition.

Figure 3. Remote chemomagnetic modulation of mouse behaviour using chemogenetics.

a, A photograph and a schematic of the forced swimming test (FST) assay within an AMF coil. b, The mouse VTA was situated within the region of uniform AMF by adjusting the swimming tank water level. The color map represents the cross sectional view of the magnetic flux distribution as calculated by a finite element model for the AMF coil. c, Classification of mouse baseline mobility to identify adaptation in FST. Inset: the mobility percentage during the 6 min FST for all tested mice. The shaded areas display the Gaussian distribution of mouse mobility percentage on each day. Blue: training day, Day 0. Red: test days, Day 1–3. n represents the number of test trials. d, Averaged motion energy curves for mice undergoing FST. The energy is calculated from the pixel changes in each frame of the FST videos. Solid lines: mean, shaded areas: s.e.m. AMF conditions: H₀ =45±2 mT, ƒ = 164 kHz. The blue area represents AMF exposure, while the grey area indicates the absence of an AMF. n represents the number of subjects and test trials. e, Mice expressing hM3D(Gq) in the VTA neurons (hM3D(Gq)⁺) and injected with CNO-loaded magnetoliposomes (CNO⁺) exhibit enhanced swimming in response to the AMF stimulus (mean ± s.e.m., two-way ANOVA and Tukey’s multiple comparisons test, F_{3, 122} = 5.387, ** 0.001≤p< 0.01, **** p<0.0001). pre: pre-stimulus epoch. dur: during AMF stimulation (or no AMF stimulation) epoch. post: post-stimulus epoch. Each marker represents an FST trial and n represents the number of trials. f, Repeated enhancement of swimming behaviour is observed in hM3D(Gq)⁺, CNO⁺ group in response to AMF stimulation (two-way ANOVA and Tukey’s multiple comparisons test, F_{4, 36} = 0.05789, n.s. p≥0.05, * 0.01≤p<0.05, ** 0.001≤p<0.01). n represents the number of subjects and test trials.

Figure 4. Remote chemomagnetic modulation mediated by endogenous receptors.

a, Top: The experimental scheme for the mouse social preference test with an AMF coil encompassing the middle chamber. The shaded radial area within the test chambers (90% of the chamber length/width) was defined as the close interaction zone. Bottom: A representative heat map tracing the position of a mouse in social subject and novel object chambers during the preference test. b, The ratio of time spent in the social interaction chamber to the object (neutral) chamber is compared for mice subjected to AMF. The group with agonist-loaded magnetoliposomes exhibits enhanced social preference following exposure to AMF (mean ± s.e.m., two-way repeated measures ANOVA and Sidak’s multiple comparisons test, close interaction F_{3, 113}= 3.053, entire chamber F_3,113=3.547, **** p<0.0001). n represents number of trials. c, The percentage of close interaction in the social chamber. The group injected with agonist-loaded magnetoliposomes spent more time in the close interaction zone (mean ± s.e.m., two-way repeated measures ANOVA and Sidak’s multiple comparisons test, F_{3, 113}= 3.122, **** P<0.0001). n represents number of trials. d, The group injected with agonist-loaded magnetoliposomes repeatedly shows increased social preference following AMF exposure (mean ± s.e.m., two-way repeated measures ANOVA and Sidak’s multiple comparisons test, F_{2, 27} = 0.5717, n.s. p≥0.05, * 0.01≤p< 0.05, ** 0.001≤p< 0.01). n represents the number of subjects and test trials.