Fig 1.
Peripheral stimulation initiates widespread EV uptake in the brain.
(A) Schematic representation of the method to map EV mRNA transfer from blood to the brain. Cre mRNA contained in blood-derived EVs is taken up by neural cells, leading to excision of the stop-loxP site and induction of marker gene expression. (B) HC from a control vav-iCre-R26EYFP mouse compared to 48 h after IP LPS injection showing frequent recombination events in the hippocampal area (C–G). Recombined cells are predominantly neurons and microglia (D–G), including DA neurons (H + I) and microglia (J + K) in the SN. Arrowheads indicate recombined cells with a neuronal morphology that are TH-negative. Both structures show similar levels of recombination. (L) Data are presented as mean ± SD. p = 0.000666 two-tailed nonparametric Wilcoxon–Mann–Whitney U test for all populations compared to zero marker-gene–positive cells in all control animals, n = 10, underlying data can be found in S2 Table. (M + N) Recombination after LPS treatment in EpoR-iCre mice is restricted to microglia. Scale bars, 500 μm in B; 100 μm in D, F, J, M; 10 μm in E, G, I; 50 μm in H; 25 μm in K; 5 μm in N. DA, dopaminergic; DG, dentate gyrus; EV, extracellular vesicle; EYFP, enhanced yellow fluorescent protein; HC, hippocampus; IP, intraperitoneal; LPS, lipopolysaccharide; SN, substantia nigra; TH, tyrosine hydroxylase; YFP, yellow fluorescent protein.
Fig 2.
Neuronal activation by proteasome inhibition is sufficient to trigger EV uptake in DA neurons.
(A) Unilateral infusion of the selective proteasome inhibitor epoxomicin into the ventral midbrain leads to increased in vivo firing frequencies of DA SN neurons. Two weeks after infusion, no marker-gene–expressing DA neurons were discernible in the contralateral hemisphere (B). In contrast, DA neurons in the ipsilateral hemisphere frequently initiated marker gene expression (C and D). (E) Percentages of marker-positive DA neurons in the SN (average of n = 4 ± SD. p = 0.0571 two-tailed nonparametric Wilcoxon–Mann–Whitney U test compared to zero marker-gene–positive cells in control animals, n = 3). Underlying data can be found in S2 Table. Scale bars, 100 μm in B, C; 10 μm in D. CC, corpus callosum; CE, cerebellum; CTX, cortex; DA, dopaminergic; DG, dentate gyrus; EV, extracellular vesicle; HC, hippocampus; LV, lateral ventricle; MB, midbrain; OB, olfactory bulb; SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; TH, tyrosine hydroxylase; VTA, ventral tegmental area; YFP, yellow fluorescent protein.
Fig 3.
Neuronal activation by optogenetic stimulation.
(A) Scheme for the injection of ChR2-mCherry-AAV in layer 5 of the primary visual cortex and optical stimulation. (B) Expression pattern of ChR2-mCherry in primary visual cortex (V1) 4 weeks after AAV injection. Layer-specific expression of ChR2-mCherry, mainly in layer V of V1. (C) Membrane-bound expression of ChR2-mCherry. (D) Representative in vivo LFP traces from mouse 4 upon optic-fiber–based illumination with blue light with an intensity of 16 and 191 mW/mm2 at the tip of the fiber. (E) Normalized LFP amplitudes plotted against light intensity. Averaged amplitude was fitted into a nonlinear curve. The dotted line marks the light intensity used in subsequent experiments (approximately 185 mW/mm2). Underlying data can be found in S2 Table. (F) Control sections of AAV-injected mice without optical stimulation as well as from mice that received only an optical stimulation without AAV injection (G) (the white arrowhead indicates marker-gene–positive meningeal macrophages). (H) Recombination occurs in ChR2-mCherry–positive and negative neurons and also in the HC (arrowheads) (H–K) but only in a few Iba1-positive microglia (L). Projections of ChR2-expressing neurons extend to the contralateral side and recombination can also be observed in target areas (M + N). No marker-gene–positive cells can be observed in sections from the forebrain (O) or the cerebellum (P). Scale bars, 500 μm in B, H, P; 50 μm in C; 100 μm in F, G, I, M; 10 μm in J, N; 5 μm in L; 20 μm in K; 1 mm in O. AAV, Adeno-Associated Virus; ChR2, channelrhodopsin-2; Clb, calbindin; HC, hippocampus; LFP, local field potential; YFP, yellow fluorescent protein.
Fig 4.
Neuronal stimulation by novel object placement.
(A) Novel objects were placed in mouse cages and left for 1 hour. Brains were analyzed along their entire rostrocaudal length 48 h thereafter (B). No marker gene expression was observed in controls in contrast to placement mice (C). Recombination occurred mainly in neurons in hippocampal areas DG and CA1 and CA2 but also in some microglia (D–F). Marker gene expression was absent or very rare in other brain areas such as forebrain (G), hindbrain (H), and cerebellum (I). A single marker-gene–positive neuron in the hindbrain is shown in inset (H). Scale bars, 100 μm in C, D, E; 500 μm in H, I; 10 μm in F and inset in H; 1 mm in G. Calb, calbindin; Cb, cerebellum; DG, dentate gyrus; FB, forebrain; HB, hindbrain; MB, midbrain; YFP, yellow fluorescent protein.