Abstract
The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4–8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.
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Change history
09 November 2016
Minor changes were made to Figs 4, 5 and Extended Data Fig. 8.
17 November 2016
The resolution of Fig. 4 was increased.
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Acknowledgements
We thank D. Franzen, G. Jäger, T. Simon, V. Staiger, H. Tultschin and F. Voss for technical support, and A. Lepier for viral vector expertise. M. Sperling, P. Goltstein and A. Grade helped with hardware and software. This work was supported by the German Research Foundation (SFB 870 ‘Neuronal Circuits’: M.G., L.D., K.-K.C., T.B. and M.H.; SPP 1757: M.G. and L.D.), the Advanced ERC grant ChroNeuroRepair (M.G.), the Helmholtz Alliance Icemed (M.G.), the Boehringer Ingelheim Fonds (S.F.), and the Max Planck Society (S.F., T.B. and M.H.).
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Contributions
The original idea for the study came from M.G. M.G, M.H. and T.B. then initiated the study and planned the experimental approach. S.F., S.G., M.G., M.H. and L.D. designed the experiments; S.F. performed in vivo imaging experiments and analysis; S.G. performed the experiments in fixed tissue including connectivity experiments and analysis. M.G. and L.D. provided the lesion model. K.-K.C. provided the rabies virus and expertise for its use for monosynaptic tracing. Finally, M.G., M.H., S.G. and S.F. wrote the paper with input from T.B., L.D. and K.-K.C.
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Extended data figures and tables
Extended Data Figure 1 Lesion model and identity of transplanted neurons.
a, Left, accumulation of Ce6 rhodamine beads (red) around neuronal cell nuclei (NeuN, white) in V1 contralateral to the beads injection. Right, high-magnification panels with DAPI+ (blue) nuclei, not all of which contain red fluorescent beads, but those that do are NeuN+ neurons (arrowheads). b, TUNEL staining (red) performed 3 days after laser illumination overlaps with loss of NeuN immunoreactivity (white), indicating degenerating neurons, and accumulation of condensed apoptotic-like nuclei (DAPI, blue; inset shows high magnification). c, Z-stack projection (left) and examples of cells in single optical sections (right) shows that most transplanted neurons express Cux1 (white; arrowheads, Cux1+ cells; arrow, Cux1− cell). Scale bars, 50 μm (a, left, b), 20 μm (a, right), 25 μm (c).
Extended Data Figure 2 Identity of embryonic neurons before transplantation.
a, In vitro Cux1 staining (white) on acutely dissociated cells from E18.5 cortex previously labelled by in utero electroporation at E14.5 with an RFP-expressing plasmid (red). b, c, Insets shown in a. Green arrowheads show examples of Cux1− cells (nuclear staining in blue, DAPI), which validate the specificity of the immunolabelling; yellow arrows highlight examples of Cux1+ cells, which are the majority among RFP+ neurons. Scale bars, 50 μm (a) and 20 μm (b, c).
Extended Data Figure 3 Intrinsic signal imaging and location of grafting site, neuron reconstruction, and control for fusion events.
a, Top, overlay of visual stimulus-evoked intrinsic signal (colour-coded in green) and the blood vessel pattern through a cranial glass window above V1. Red dotted line shows area of laser photoactivation. Bottom, wide-field fluorescence image through the same cranial window (V1, green dotted line). Grafting site (GFP+, white arrowhead) in the binocular region of V1. b, In vivo two-photon z-stack projection (inverted) of a grafting site at 52 dpt, top and side view. c, Left, in vivo two-photon z-stack projection of a grafting site at 45 dpt (inverted; same as in Fig. 1c). Right, reconstructions (skeleton) of example neurons present in the grafting site depicted on the left reveals typical layer 2/3 like morphology. Apical dendrites (magenta) branch from one prominent main dendritic trunk and extend to the surface. Basal dendrites (green) extend from the cell body and reach 300 μm below the pial surface. Top row, top view; bottom row, side view. d, Genetic strategy to control for fusion events. Emx1-Cre-driven GFP+ donor cells were transplanted into tdTomato reporter mice (n = 10). e, Absence of tdTomato fluorescence in GFP+ grafted neurons in vivo. Line plot (along the white dotted line) across a GFP+ example neuron (green arrowhead) shows that the neuron is tdT negative. Rhodamine beads (red arrowheads) are equally detected in both channels. Units, 8-bit greyscale. Scale bars, 100 μm (a, b), and 50 μm (c, e).
Extended Data Figure 4 Modes of formation of spines and boutons, and long-term survival of grafted neurons.
a, Dendrites extend and arborize considerably before the first spines form on bare dendrites. Example of a naked dendrite bare from 4 dpt (not shown) to 8 dpt that forms the first spines 12 dpt (empty green arrowheads indicate the location on the dendrite and filled green arrowheads indicate the newly formed spines). Two spines remain stable until 35 dpt (blue arrowheads). b, Bouton formation precedes spine formation. Example axon at 4 dpt; arrowheads (green) indicate new boutons that remain stable over subsequent time points (blue arrowheads). c, Boutons are able to form within a few micrometres of the axonal growth cone (green star). Individual boutons that have formed in the vicinity of a growth cone are able to survive for days and weeks (blue arrowheads). d, Grafted neurons that survived the early phase of integration (>12 dpt) remained stable until the end of the experiment (here 10 months) and probably for the rest of the animals’ life. e, f, Comparison of early- and late-formed spines and boutons with structures formed at 4–9 wpt. Median survival ratios indicate the relative survival. Hazard ratios indicate the relative chance for structures to be lost. e, Early formed dendritic spines (<12 dpt) have a 1.5–2.4 times higher chance of being eliminated compared to spines newly formed at 4–9 wpt. Hazard ratios remain increased (>1.2) up to 24 dpt. Spines formed at 9 mpt, however, have a very high chance of survival. Although the median survival at 4–9 wpt is 28 days, more than half of the spines formed at 9 mpt survive at least for 51 days (blue triangle, arbitrary value as survival is >50%). f, Early formed axonal boutons (<7 dpt) are 2–8 times more likely to be eliminated compared to boutons newly formed at 4–9 wpt. Boutons formed at 9 mpt, have a 4 times higher chance of survival. Scale bars, 10 μm (a–c) and 20 μm (d).
Extended Data Figure 5 Local and brain-wide monosynaptic input to transplanted neurons.
a, Examples of the transplantation site in three different animals selected to demonstrate that RFP+ transplanted neurons (left to right: many to few) are consistently surrounded by a large number of GFP-only labelled connecting neurons from the host. b, 3D diagram of the brain-wide monosynaptic input connectome for each of the examples in a. Shown are the location of starter neurons in V1 (yellow) and the innervating neurons (green), either local (green circle) or distant (green lines, thickness of the line represents the connectivity ratio for a given area and respects the ranges displayed in Fig. 4f). Note the strong input from thalamic nuclei, in particular the dLGN, in all individual cases. Importantly, the number of areas and of input neurons in a given area correlates with the number of starter neurons in V1 (see Extended Data Fig. 7). Schematics of brains taken from Brain Explorer, Allen Institute for Brain Science. c, Distribution of neurons providing synaptic input to transplanted neurons, example traced at 4 wpt. Number of GFP-only (green) and GFP/RFP double-labelled (yellow) cells throughout the transplanted hemisphere. Each bar corresponds to one coronal section, from posterior (left) to anterior (right) coordinates of the mouse brain. Sections including the visual cortex are highlighted in grey. Note the overrepresentation of local connections compared to long-range projections.
Extended Data Figure 6 Normal circuitry of upper layer neurons in V1.
a, Schematic depicting experimental procedure. At E15.5, L2/3 neuronal progenitors were in utero electroporated to express RFP-G-TVA or RFP-TVA. Electroporation was targeted to the somatosensory cortex to achieve a small number of transduced cells in the nearby visual cortex. Subsequently, in adult mice, ΔG-GFP (EnvA) RABV was injected into V1 of the in utero electroporated hemisphere. b, Sagittal section stained with DAPI (blue) shows RFP-G-TVA electroporated neurons (red) restricted to the upper layers. Axon collaterals of these neurons cause dimmer red labelling in layer 5. In green, pre-synaptic neurons in and around the area of RABV injection and transduction of few RFP-G-TVA (red) cells. c, Higher power micrographs (left) showing RABV-traced cells (green) in V1 with RFP-G-TVA-expressing (top) or RFP-TVA-expressing (bottom) upper layer neurons (red). Right, high magnification shows that a modest number of starter cells (arrows; GFP/RFP) connects robustly with local neurons, while, if primarily infected cells lack G, only occasional and local GFP-only cells are observed (arrowheads). Scale bars, 1 mm (b), 100 μm (c, left) and 50 μm (c, right).
Extended Data Figure 7 Quantitative analysis of the monosynaptic synaptic tracing of regenerated and endogenous neuronal circuits.
a, Connectivity ratio for each anatomical area, obtained from RABV tracing at 4 and 12 wpt, or in control mice, and calculated as described in Methods. The number of transplants/mice with synaptic input from a given area is specified (from a total of 6 mice per group, or 3 control mice in the endogenous circuit tracing). The data indicate that some areas project few axons to V1, and thus a higher number of starters results in increased probability of synapse formation, necessary to unveil these connections; while other areas project massively to V1 and are therefore traced even from only one starter neuron in V1 (Vis, dLGN). The number of starter neurons varied across mice owing to variability of the number of transplanted cells and efficacy of RABV injection (number of starter neurons in each of the 6 mice at 4 wpt: 1, 3, 16, 24, 32 and 80, and in each of the 6 mice at 12 wpt: 1, 1, 3, 10, 13 and 29). Blue shading indicates connections formed only late (12 wpt), grey shading marks the ones that did not form. b, Top, 3D diagram of the brain-wide input connections at 4 wpt (green, n = 6) and additional connections revealed at 12 wpt (blue, n = 6; regions shown in the bottom diagram, rendered in Brain Explorer 2, Allen Institute for Brain Science.). Shown are the location of starter neurons in V1 (yellow) and the innervating neurons (green/blue), either local (green circle) or distant (green/blue lines; thickness of the line represents the connectivity ratio for a given area and respects the ranges displayed in Fig. 4f). Note the pronounced input from visual cortex and dLGN. c, RABV-traced inputs to transplanted neurons observed exclusively at 12 wpt, all known to project to V1 (for dorsal striatum see also online Allen Mouse Brain Connectivity Atlas, experiment 112458831, section 90; experiment 100142580, section 90; experiment 112307754, section 82). Sagittal brain sections with nuclear staining (DAPI) indicate the field magnified below (red square). Yellow text corresponds to regions where GFP-only neurons were observed, and white text denotes nearby regions for anatomical reference. Individual cells are shown at high magnification below. For abbreviations see Extended Data Table 1. Scale bars, 200 μm (c) and 50 μm (insets).
Extended Data Figure 8 Transplanted neurons in V1 receive topographically organized inputs from the dLGN.
a, b, Examples of dLGN-containing clusters of cells innervating transplants at distinct locations in V1 (mouse 1, 2 and 3), revealed by RABV retrograde tracing. a, Each column shows three consecutive sections from medial to lateral (M–L); indicated is the distance to the medial boundary of the dLGN. Dashed lines indicate the outline of the dLGN, dorsal is up, anterior is to the right. b, 3D reconstruction of V1 starter cells and their pre-synaptic dLGN cells from mice 1, 2 and 3, plotted into the same rendered V1 (top view) and dLGN (lateral, top and anterior views), and shown in a different colour for each mouse (each point represents one cell). All three clusters receive inputs from topographically corresponding parts of the dLGN. c, 3D reconstruction comprising all mice (n = 8) analysed shows that pre-synaptic cells in the dLGN always cluster, that cluster size in V1 and dLGN correlates, and the progressive nature of the topography (see intermediate clusters, for example, pink and orange in ML–ML correlation). Tilted latero-anterior view of V1 demonstrates the similar depth of each transplant in V1, reaching down to approximately the middle of the cortical thickness. d, Correlation between the extent of labelling in V1 (starter neurons) and the respective dLGN (pre-synaptic neurons), by quantification either of the total number of cells (left) or of slices containing cells as a measure for cells’ dispersion in the cluster (right), at 4 (light grey) and 12 wpt (dark grey). e, Top, example of the 3D boundary encompassing a cluster of transplanted cells in V1 and the corresponding pre-synaptic cell cluster in the dLGN. Blue dots represent individual cells, red circle indicates the centroid of each cluster. Bottom, centroid coordinates of V1 and dLGN cell clusters (blue dots) are plotted relative to the centroid of the V1/dLGN rendered volumes (x, y, z = 0, 0, 0; intersection of the red lines), for each two dimensions in space (A–P, antero-posterior; M–L, medio-lateral; D–V, dorso-ventral). Orange dots show results from analysis of the native circuitry. Regression line and coefficient of determination (R2; values at top right corner) for the experimental group are indicated for each plot; blue lines/values indicate slopes significantly non-zero, that is, data close to the fitted linear regression (P < 0.05); grey lines/values indicate non-significant correlations. Regarding dLGN-to-V1 projections, the dimensions AP–AP, DV–AP and ML–ML inversely correlate, as is known and confirmed in our control data (orange dots). Also, a DV–DV correlation is revealed, although it probably results from overrepresented DV coordinates of cells in V1 due to the V1 curvature. Scale bar, 100 μm.
Extended Data Figure 9 Responses to visual stimulation: boutons on the same axon, binocular responses, distribution of preferred directions.
a, Left, axon of a transplanted neuron (tdTomato+) expressing GCaMP6, single optical plane, maximum projection of all frames of one stimulation sequence (see Methods). Right, individual (grey) and average (blue) responses (arbitrary units) of 4 boutons (indicated on the left) to visual stimulation with gratings moving in 8 directions (grey bars, direction indicated on top). Note highly similar responses of all boutons. b, Individual transplanted neurons respond to ipsilateral (IL) and contralateral (CL) eye stimulation. Top, polar plots; bottom, single plane, maximum projection of all frames of one stimulation sequence. c, Transplanted neurons are tuned to all directions, with a slight overrepresentation of cardinal directions. Cumulative absolute number of grafted neurons across imaging time points, sorted according to their respective preferred direction. Scale bars, 5 μm.
Extended Data Figure 10 Normalized average 2D tuning recorded from example neurons, axons and spines.
a, b, Normalized average tuning plots ± s.e.m. for the examples presented in Fig. 5f, g (a) and in Fig. 6a, b (b). Same arrangements and time points as in Figs 5 and 6.
Extended Data Figure 11 Orientation and direction selectivity assessed with Gaussian fits.
a, Example cell displaying strong orientation selectivity (right, polar plot), fitted with a double Gaussian (DG) function. b, Example cell displaying strong direction selectivity (right, polar plot), fitted with a single Gaussian (SG) function. c, Individual DG fits of 17 cells in total (n = 4 mice) at 6, 9 and 15 wpt. d, Individual SG fits of 17 cells in total (n = 4 mice) at 6, 9 and 15 wpt. e, f, Peak amplitudes are constant and do not correlate with specificity. e, Consistent average peak amplitudes across imaging time points. P = 0.6932 (not significant), Kruskal–Wallis test. f, Orientation and direction selectivity indices do not correlate with average peak amplitudes (OSI, R2 = 0.0692; DSI, R2 = 0.1495; dashed lines, 95% confidence interval). Reported48 nonlinearity effects of calcium indicators would predict higher selectivity indices at lower average peak amplitudes.
Supplementary information
Morphological development of a transplanted neuron
Neuron 3 to 92 dpt (same as in Fig. 1), developing a L2/3 pyramidal cell-like morphology within 3 wpt. Stable overall morphology 4 to 13 wpt. In vivo two-photon z-stacks depicted as maximum projections, time series. (MOV 2103 kb)
Formation of dendritic spines
Dendrite 6 to 75 dpt, forming first dendritic spines at 9 dpt. Z-stack maximum projections, time series. (MOV 246 kb)
Formation of axonal boutons
Axon 5 to 84 dpt (same as in Fig. 2), forming first axonal boutons at 5 dpt, a secondary branch forms at 7 dpt. Z-stack maximum projections, time series. (MOV 340 kb)
3D reconstruction of dLGN relay cells retrogradely traced in mice with distinct transplantation sites in V1
Data from each mouse (n=8) is represented with a distinct color, and each sphere corresponds to one neuron (n=3-96/mouse). Animation starts with an anterior view and then rotates around several axes, demonstrating that cells form segregated clusters in specific parts of the dLGN (surface in wireframe; see Extended Data Fig. 8c). (MOV 9097 kb)
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Falkner, S., Grade, S., Dimou, L. et al. Transplanted embryonic neurons integrate into adult neocortical circuits. Nature 539, 248–253 (2016). https://doi.org/10.1038/nature20113
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DOI: https://doi.org/10.1038/nature20113
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