Stable long-term chronic brain mapping at the single-neuron level

Journal name:
Nature Methods
Volume:
13,
Pages:
875–882
Year published:
DOI:
doi:10.1038/nmeth.3969
Received
Accepted
Published online

Abstract

Stable in vivo mapping and modulation of the same neurons and brain circuits over extended periods is critical to both neuroscience and medicine. Current electrical implants offer single-neuron spatiotemporal resolution but are limited by such factors as relative shear motion and chronic immune responses during long-term recording. To overcome these limitations, we developed a chronic in vivo recording and stimulation platform based on flexible mesh electronics, and we demonstrated stable multiplexed local field potentials and single-unit recordings in mouse brains for at least 8 months without probe repositioning. Properties of acquired signals suggest robust tracking of the same neurons over this period. This recording and stimulation platform allowed us to evoke stable single-neuron responses to chronic electrical stimulation and to carry out longitudinal studies of brain aging in freely behaving mice. Such advantages could open up future studies in mapping and modulating changes associated with learning, aging and neurodegenerative diseases.

At a glance

Figures

  1. Syringe-injectable mesh electronics for chronic brain mapping and modulation.
    Figure 1: Syringe-injectable mesh electronics for chronic brain mapping and modulation.

    (a) Schematic showing a mouse with stereotaxically injected mesh electronics (gold) bonded through conductive ink (black lines) printing to a flexible flat cable (FFC, red arrow), which is folded afterwards to minimize its profile. Inset, a zoomed-in view of the blue dashed box illustrating seamless integration of the mesh electronics with neural network. The gold lines and the white and yellow circles represent metal interconnects and recording and stimulation electrodes, respectively. (b) Microcomputed tomography (micro-CT) image showing a lateral view of a mouse head with injected mesh electronics (blue dashed box) and folded FFC (red arrow). Axes labeled with A, P, D and V represent anterior, posterior, dorsal and ventral anatomical directions, respectively. Scale bar, 2 mm. (c) Picture showing the conductive ink (black lines) printing process to electrically connect the mesh electronics (yellow arrow) to the FFC (red arrow) during surgery. The white arrow highlights the implantation site. (d) Picture showing a freely behaving mouse with mesh electronics injected and FFC attached (red arrow). The width of the FFC in c and d is ~8 mm.

  2. Long-term stable recording without signal degradation over six months and immunohistochemistry staining of mesh electronics-brain tissue interface.
    Figure 2: Long-term stable recording without signal degradation over six months and immunohistochemistry staining of mesh electronics–brain tissue interface.

    (a) Representative 16-channel local field potential (LFP; heat maps) with amplitudes color-coded according to the color bar on the far right and single-unit spike (traces) mapping from the same mouse at 2 (left) and 4 (right) months postinjection. The x-axes show the recording time while the y-axes represent the channel number of each recording electrode with relative position marked by red dots in the schematic (leftmost panel). (b) Time evolution of average spike amplitudes of representative channels from four different mice. Mouse 1 represents the recordings shown in a, with Channels A and B denoting channels 10 and 3, respectively. Mouse 2–Channel A, Mouse 2–Channel B and Mouse 3–Channel A were used for analyses shown in Figures 3 and 5 and in Supplementary Figures 10 and 12. Mouse 4–Channel A represents the recordings shown in Supplementary Figure 6. (c) Time-dependent impedance values at 1 kHz of the channels shown in b. (d) Immunohistochemical staining images of horizontal brain slices at 2 (left, hippocampus (HIP)), 6 (middle, cortex (CTX)) and 12 weeks (right, CTX) postinjection. Red, yellow and blue colors correspond to neurofilaments, NeuN and mesh electronics, respectively. Scale bars, 100 μm. (e) Neurofilament (red), NeuN (yellow), GFAP (cyan) and iba-1 (purple) fluorescence intensity normalized against background values (gray dashed horizontal lines) plotted versus distance from the interface. The pink-shaded regions indicate the interior of the mesh electronics. All error bars in this figure reflect ±1 s.e.m.

  3. Consistent tracking of the same group of neurons.
    Figure 3: Consistent tracking of the same group of neurons.

    (a) Time evolution of representative single-unit spikes of Mouse 2–Channel A shown in Figure 2 clustered by principal component analysis (PCA) over 8 months postinjection. The x- and y-axes denote the first and second principal components, respectively, and the z-axis indicates postinjection time. The color scale bars show the corresponding postinjection time points from 5 to 34 weeks (8 months) of the 3D PCA plots. (b) Timecourse analysis of average spike waveforms from each PCA cluster shown in a. (c) Time evolution of interspike interval (ISI) histograms of each of the three neurons identified in a from 3 to 34 weeks. Bin size, 20 ms. (d) Scatter plot with analysis of variance (ANOVA) of the firing parameter (n = 32 for each neuron), λ, obtained by fitting each ISI distribution profile shown in c to an exponential decay. (e) Polar plots showing the phase locking of single-unit spikes to theta oscillations (4–8 Hz) of LFPs in HIP for each of the three neurons in a at 3 and 34 weeks. (f) Scatter plot with ANOVA test of the locked phase angle (n = 32 for each neuron). For d and f, the open rectangles and bars indicate 25/75 and 0/100 percentiles, respectively. **** indicates P value < 0.0001.

  4. Multisite and multifunctional mesh electronics.
    Figure 4: Multisite and multifunctional mesh electronics.

    (a) Schematic and (b) photo showing two mesh electronics (white arrows) injected into different brain regions (motor CTX of the right cerebral hemisphere and HIP of the left hemisphere) of the same mouse. The two mesh probes were bonded to the same FFC (red dashed box in b). The width of the FFC in b is ~8 mm. (c) Multiplexed LFP and (d) single-unit recordings along with sorted spikes from the motor CTX (red traces) of one hemisphere and the HIP (blue traces) from the contralateral hemisphere. The three columns correspond to data recorded at 2 (left), 3 (middle) and 4 (right) months postinjection. The red and blue arrows in c highlight the channels corresponding to the representative spike trains shown in d. (e) Photograph showing typical mesh electronics before releasing from substrate with unipolar stimulation electrodes (black arrow) and recording electrodes (red arrows). Scale bar, 200 μm. Inset, zoomed-out photograph with yellow dashed box representing the area of e. Inset scale bar, 1 mm. (f) Peristimulus raster plot showing spike events (black ticks) of 150 stimulation trials (red solid line, stimulation pulse). Inset, representative recorded spike trains from −0.125 to 0.875 s. The red arrow indicates the stimulation pulse. (g) First-spike latency distributions of stimulus-evoked firings recorded from two different electrodes (channels 1 and 2) located in the same cerebral hemisphere but with progressively increasing distance from the stimulation electrode at 4, 6 and 14 weeks postinjection. Spike sorting and PCA clustering results are displayed as insets. The color of each sorted spike cluster corresponds to that of each PCA group.

  5. Longitudinal study of brain aging at the single-neuron level.
    Figure 5: Longitudinal study of brain aging at the single-neuron level.

    (a,b) Time evolution of average spike firing rate (I) and average peak-to-trough time τ (defined in upper left inset in a, II) with average spike waveforms shown as bottom-right insets (II) of each neuron identified from the PCA clusters in Supplementary Figure 12 from representative channels of Mouse 2 (a) and 3 (b), respectively. The x-axes show the corresponding mouse ages in all panels. The error bars in I show fitting errors, and * indicates statistically significant (P < 0.05, double-sided t-test, n = 50) decrease of firing rate compared with that at age of 48 weeks. The error bars in II show ± 1 s.d.

  6. Chronic recordings from a freely behaving mouse.
    Figure 6: Chronic recordings from a freely behaving mouse.

    (a) Photograph of a typical freely behaving mouse recording. Voltage amplifier was directly positioned near the mouse head to minimize mechanical noise coupling. A flexible serial peripheral interface (SPI) cable was used to transmit amplified signals to the data acquisition systems. Inset, a zoomed-in view showing the conductive ink (black lines), FFC (red arrow), Omnetics connector (yellow arrow) and the voltage amplifier (blue-green rectangle). The width of the voltage-amplifier is ~1.5 cm. (b) Single-unit spike recordings at 5 weeks postinjection from five representative channels, two of which (Channels D and E, shown in red) are located in the somatosensory CTX, when the mouse was whisking food pellets (I) and foraging in the cage (II). (c) Bar charts summarizing the changes in firing rate for the same five channels shown in b during whisking (black bars) and foraging (white bars) at 5 (top), 10 (middle) and 27 weeks (bottom) postinjection. The two channels (Channels D and E) with whisking-associated neuronal responses are highlighted with red borders. Error bars indicate ± 1 s.e.m. (d) Polar plots showing phase locking of single-unit spikes recorded in the CTX barrel field (Channel D) to the theta oscillations (4–8 Hz) of LFPs in the HIP (Channel A) when the mouse was whisking (left column) and foraging (right column) at 5, 10 and 27 weeks.

  7. Schematic steps of mesh electronics fabrication.
    Supplementary Fig. 1: Schematic steps of mesh electronics fabrication.

    Components include silicon wafer (light green), nickel relief layer (dark green), polymer ribbons (blue), metal interconnects (black) and exposed metal electrodes (red). For each step (a-g) both top and side views are shown, where the side view corresponds to a cross-section taken at the position indicated by the white horizontal dashed line in the top view image of (a). (h) Zoomed-in views of regions highlighted by black (exposed Pt electrodes) and red dashed boxes (fully passivated interconnects) in (g).

  8. Schematic structure of mesh electronics.
    Supplementary Fig. 2: Schematic structure of mesh electronics.

    (a) Schematic of the injectable mesh electronics. Blue lines highlight the overall mesh structure and indicate the regions of supporting and passivating polymer layers, the ~horizontal orange lines indicate Au metal interconnects between input/output (I/O) pads (orange filled circles, indicated by the magenta arrow) and Pt recording electrodes (green filled circles), respectively. The solid-line black box at left highlights several of the recording electrodes, and the dashed-line black box in the middle highlights several of the metal interconnects of the mesh electronics. (b) A zoomed-in view of the recording electrodes highlighted by solid-line black box in (a). (c) A zoomed-in view of the mesh electronics highlighted by dashed-line black box in (a). (d) A zoomed-in view of a unit cell of the mesh (dashed-line green box in (c)) with the same color codes as in (a, c). Polymer ribbons (blue) with and without metal interconnects (orange) correspond to longitudinal and transverse elements, respectively. L1 and L2 show spacing between longitudinal and spacing between transverse elements, respectively; W1 and W2 are widths of the longitudinal and transverse mesh elements, respectively; and Wm is the width of metal interconnect lines.

  9. Schematics illustrating method used to load the mesh electronics into a glass capillary needle and subsequently inject into a medium.
    Supplementary Fig. 3: Schematics illustrating method used to load the mesh electronics into a glass capillary needle and subsequently inject into a medium.

    (a) The injectable mesh electronics (dark blue) suspended in solution (light pink) was drawn into the glass needle such that the I/O pads (yellow dots, indicated by green arrows) of the mesh enter the tube first. (b) After the mesh electronics was fully loaded into the glass needle with mesh end at needle tip (magenta dashed box), the needle was removed from the solution. (c) The glass needle was mounted in an x-y-z manipulator for injection into solution, gel/polymer or tissue (light blue). Black arrows indicate the direction of the fluid flow during loading and injection.

  10. Mesh electronics injection and chronic recording from mouse brain.
    Supplementary Fig. 4: Mesh electronics injection and chronic recording from mouse brain.

    (a) Image of a mouse fixed in a stereotaxic frame with scalp skin retracted, and a hole drilled through the skull plate. The glass needle (yellow arrow) loaded with mesh electronics is visible directly above the skull in this image. A flexible flat cable (FFC) is visible at the left of the image supported on a ceramic scaffold. A 0-80 grounding screw (white arrow) was positioned at the posterior side of the skull. (b) Image showing the relocation of the I/O end of the post-injected mesh electronics (red arrow) towards the FFC. (c) Image post-injection into the brain showing the input/output (I/O) region of the mesh electronics unfolded onto the FFC. Inset shows a zoom-in view of the red dashed box, which highlights the unfolded I/O part (red arrow) of the injected mesh electronics. (d) Image representing the unfolded I/O pads of the mesh electronics which were electrically-connected to the FFC using the conductive ink printing process described in ref. 33. The red dashed box highlighted by the inset shows the details of the bonding with the red and cyan arrows indicating the mesh electronics and conductive ink connections, respectively. (e) Image showing the FFC fixed on top of the mouse skull using dental cement. (f) Image of a fully awake but restrained mouse during chronic in vivo brain recording. The white and black arrows highlight the grounding screw and the connector between FFC and external recording setups, respectively. See Online Methods for all experimental details of surgery and injection. The width of FFC in all panels is ~8 mm.

  11. Correlation maps of chronic multiplexed brain recording.
    Supplementary Fig. 5: Correlation maps of chronic multiplexed brain recording.

    (a,b) Correlation maps of 16-channel local field potential (LFP) recordings at two (a) and four (b) months post-injection. (c,d) Correlation maps of 16-channel extracellular action potential recordings at two (c) and four (d) months post-injection. Colors indicate the correlation coefficient between any two given channels according to the color bar shown on the far right. All the maps were calculated from 2 s long data traces at both time points. See Online Methods for details of correlation coefficient calculations.

  12. Long-term stable recording without signal degradation over six months.
    Supplementary Fig. 6: Long-term stable recording without signal degradation over six months.

    (a) Representative extracellular action potential recordings from the same electrode (Mouse4-ChannelA shown in Fig. 2b) located in somatosensory cortex (CTX) at different time points post-injection. (b) Spike sorting results of the corresponding recordings shown in (a). All of the spikes from the corresponding time points in (a) are included with ca. 50 spikes at each point. (c) Autocorrelation histograms of average waveforms of sorted spikes from 30 1-min segments within a 30-min recording session for Mouse4-ChannelA at 3 months post-injection. (d) Autocorrelation histograms of average waveforms for each of the two identified clusters (color coded in magenta (neuron 1) and green (neuron 2)) for Mouse4-ChannelA at the 8 time points shown in (b). See Online Methods for details of waveform autocorrelation calculations. (e) Time evolution of normalized average peak-to-peak spike amplitudes across all channels with single-unit action potentials recorded (the average was done across 14, 5, 6 and 7 channels for Mouse 1 to 4, respectively) from four mice. The spike amplitude was normalized (value=1.0, gray dashed horizontal lines) against the average peak-to-peak amplitude values between 5 to 26 weeks post-injection for each channel. (f) Average impedance values at 1 kHz across all channels with recorded single-unit action potentials of the four mice shown in (e) plotted as a function of time over the same time period. The shaded areas in (e) and (f) indicate ±1 standard error of the mean (s.e.m.).

  13. Immunohistochemical images of horizontal sectioned mouse brains containing the mesh electronics from different times post-injection.
    Supplementary Fig. 7: Immunohistochemical images of horizontal sectioned mouse brains containing the mesh electronics from different times post-injection.

    (a) GFAP stained images of representative horizontal brain slices used for normalized fluorescence intensity plots (Fig. 2e) showing the interfaces between mesh electronics and astrocytes, and (b) iba-1 stained images of representative horizontal slices used for normalized fluorescence intensity plots (Fig. 2e) showing the interfaces between mesh electronics and microglia at 2, 6 and 12 weeks post-injection. All samples were 10 μm-thick. Blue, cyan and purple correspond to mesh electronics, GFAP and iba-1, respectively. Scale bars: 100 μm.

  14. Autocorrelation and cross-correlation analyses of average spike waveforms across all recording time points.
    Supplementary Fig. 8: Autocorrelation and cross-correlation analyses of average spike waveforms across all recording time points.

    (a) Auto- and cross-correlation histograms for three identified neurons of Mouse2-ChannelA from week 3 to 34 post-injection shown in Fig. 3b. (b) Auto- and cross-correlation histograms for two identified neurons of Mouse2-ChannelB from week 1 to 34 post-injection shown in Supplementary Fig. 10c. (c) Auto- and cross-correlation histograms for two identified neurons of Mouse3-ChannelA from week 1 to 26 post-injection shown in Supplementary Fig. 10d.

  15. Refractory periods of neuron firing.
    Supplementary Fig. 9: Refractory periods of neuron firing.

    (a-c) Interspike interval (ISI) histograms of the data shown in Fig. 3c at 26 weeks post-injection but replotted with a bin size of 1 ms. The data show clearly a 2-3 ms refractory period (orange-shaded regions) for neuron 1 (a), neuron 2 (b) and neuron 3 (c).

  16. Consistent tracking of the same group of neurons.
    Supplementary Fig. 10: Consistent tracking of the same group of neurons.

    (a,b) Time evolution of representative single-unit spikes of Mouse2-ChannelB (a) and Mouse3-ChannelA (b) shown in Fig. 2 clustered by principal component analysis (PCA) over eight and six months, respectively. In each plot, the x- and y-axis denote the first and second principal component, respectively, and the z-axis indicates post-injection time. (c,d) Time course analysis of average spike waveforms from each PCA cluster shown in (a) from 1 to 34 weeks post-injection (c), and for each PCA cluster shown in (b) from 1 to 26 weeks (d), respectively. (e,f) Time evolution of ISI histograms of each of the 2 neurons identified from the PCA clusters in (a) and (b) from 3 to 34 (e) and 3 to 26 weeks (f), respectively. Bin size: 20 ms. (g,h) Scatter plot with analysis of variance (ANOVA) (n=32 and 26 for (g) and (h), respectively) of the firing parameter, λ, obtained by fitting each ISI distribution profile shown in (e) and (f) to an exponential decay. All plots for Mouse3 are shown up to 26 weeks post-injection instead of 34 weeks shown for Mouse2, because Mouse3 was older than Mouse2 when the initial mesh electronics injection was carried out, and thus exhibited ageing-associated changes (shown in Fig. 5 and Supplementary Fig. 12) at an earlier post-injection time point.

  17. Rayleigh Z-test of neuron phase-locking behavior.
    Supplementary Fig. 11: Rayleigh Z-test of neuron phase-locking behavior.

    (a-c) Rayleigh Z-test of all recording data for neuron 1 (a), neuron 2 (b) and neuron 3 (c) identified for Mouse2-ChannelA from week 3 to 34 post-injection (n=32). Each trial presents the recording data at a given week. The null hypothesis is: a given neuron does not have phase-locking behavior at a given week. The majority of trials on the right side of the vertical dashed lines suggest a >95% confidence interval (p<0.05) to reject the null hypothesis.

  18. Tracking of the same individual neurons during brain ageing.
    Supplementary Fig. 12: Tracking of the same individual neurons during brain ageing.

    (a,b) Time evolution of representative single-unit spikes of Mouse2-ChannelA (a) and Mouse3-ChannelA (b) shown in Fig. 2 clustered by PCA from 35 to 57 weeks of age. In each plot, the x- and y-axes denote the first and second principal components, respectively, and the z-axis indicates the corresponding mouse age. (c,d) Representative 2D (PC1-PC2 plane) plots of the PCA results shown in (a,b) at week 52 and 57 of mouse age. (e,f) Time evolution of ISI histograms of each of the neurons identified from the PCA clusters in (a) and (b) from 35 to 57 weeks of age. Bin size: 20 ms. (g) Impedance values at 1 kHz of the two channels shown in (a-f) plotted as a function of mouse age over the same period. (h) Immunohistochemical images of a 10 μm-thick horizontal CTX brain slice showing the mesh electronics/brain tissue interface at 1-year mouse age. Red, yellow and blue colors correspond to neurofilaments, NeuN and mesh electronics, respectively. Scale bar: 100 μm. (i) Neurofilament (red), NeuN (yellow), GFAP (cyan) and iba-1 (purple) fluorescence intensity normalized against background values (gray dashed horizontal lines) plotted versus distance from the interface (see Online Methods). The pink-shaded regions indicate the interior of the mesh electronics. All error bars in this figure reflect ±1 standard error of the mean (s.e.m.).

  19. Spike recordings from a freely behaving mouse.
    Supplementary Fig. 13: Spike recordings from a freely behaving mouse.

    (a) Sorted single-unit action potentials from chronic freely behaving mouse recordings. Each column represents the sorted spikes from an individual channel shown in Fig. 6 at 5, 10 and 27 weeks post-injection. (b) Noise distributions of all red unit clusters of Channels A-E shown at week 10 in a (I), and the intrinsic recording noise of each corresponding electrode of the channel at the same week (II). See Online Methods for details of noise calculations. (c) Single-unit spikes of Channel D in (a) clustered by PCA when the mouse was actively whisking (I) and foraging (II) at week 5, 10 and 27.

  20. Autocorrelation analyses of average waveforms from week 1 to 8 post-injection.
    Supplementary Fig. 14: Autocorrelation analyses of average waveforms from week 1 to 8 post-injection.

    (a) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse2-ChannelA from weeks 1 to 8 post-injection shown in Fig. 3b. (b) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse2-ChannelB from weeks 1 to 8 shown in Supplementary Fig. 10c. Inset: Cross-correlation histogram of all raw spikes for neuron 2 between weeks 1 and 8 (light green, dashed line) and autocorrelation histogram of all raw spikes for neuron 2 on week 8 only (dark green, solid line). (c) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse3-ChannelA from weeks 1 to 8 shown in Supplementary Fig. 10d. See Online Methods for details of autocorrelation calculations.

Videos

  1. Chronic recordings from a freely behaving mouse with implanted mesh electronics probe.
    Video 1: Chronic recordings from a freely behaving mouse with implanted mesh electronics probe.
    This video shows a mouse with a head-mounted voltage-amplifier roaming freely in a cage with randomly positioned food pellets during recording. The frame rate is 25 frames per second (fps) and the video is played at 1× real time. The mesh electronics was injected into the somatosensory cortex and hippocampus, and bonded to the interface cable as described in the Online Methods.

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Author information

  1. These authors contributed equally to this work.

    • Tian-Ming Fu,
    • Guosong Hong &
    • Tao Zhou

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Tian-Ming Fu,
    • Guosong Hong,
    • Tao Zhou &
    • Charles M Lieber
  2. John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • Thomas G Schuhmann,
    • Robert D Viveros &
    • Charles M Lieber

Contributions

T.-M.F., G.H. and C.M.L. designed the experiments. T.-M.F., G.H., T.Z., T.G.S. and R.D.V. performed the experiments. T.-M.F., G.H., T.Z. and C.M.L. analyzed the data and wrote the paper. All authors discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Schematic steps of mesh electronics fabrication. (119 KB)

    Components include silicon wafer (light green), nickel relief layer (dark green), polymer ribbons (blue), metal interconnects (black) and exposed metal electrodes (red). For each step (a-g) both top and side views are shown, where the side view corresponds to a cross-section taken at the position indicated by the white horizontal dashed line in the top view image of (a). (h) Zoomed-in views of regions highlighted by black (exposed Pt electrodes) and red dashed boxes (fully passivated interconnects) in (g).

  2. Supplementary Figure 2: Schematic structure of mesh electronics. (171 KB)

    (a) Schematic of the injectable mesh electronics. Blue lines highlight the overall mesh structure and indicate the regions of supporting and passivating polymer layers, the ~horizontal orange lines indicate Au metal interconnects between input/output (I/O) pads (orange filled circles, indicated by the magenta arrow) and Pt recording electrodes (green filled circles), respectively. The solid-line black box at left highlights several of the recording electrodes, and the dashed-line black box in the middle highlights several of the metal interconnects of the mesh electronics. (b) A zoomed-in view of the recording electrodes highlighted by solid-line black box in (a). (c) A zoomed-in view of the mesh electronics highlighted by dashed-line black box in (a). (d) A zoomed-in view of a unit cell of the mesh (dashed-line green box in (c)) with the same color codes as in (a, c). Polymer ribbons (blue) with and without metal interconnects (orange) correspond to longitudinal and transverse elements, respectively. L1 and L2 show spacing between longitudinal and spacing between transverse elements, respectively; W1 and W2 are widths of the longitudinal and transverse mesh elements, respectively; and Wm is the width of metal interconnect lines.

  3. Supplementary Figure 3: Schematics illustrating method used to load the mesh electronics into a glass capillary needle and subsequently inject into a medium. (128 KB)

    (a) The injectable mesh electronics (dark blue) suspended in solution (light pink) was drawn into the glass needle such that the I/O pads (yellow dots, indicated by green arrows) of the mesh enter the tube first. (b) After the mesh electronics was fully loaded into the glass needle with mesh end at needle tip (magenta dashed box), the needle was removed from the solution. (c) The glass needle was mounted in an x-y-z manipulator for injection into solution, gel/polymer or tissue (light blue). Black arrows indicate the direction of the fluid flow during loading and injection.

  4. Supplementary Figure 4: Mesh electronics injection and chronic recording from mouse brain. (247 KB)

    (a) Image of a mouse fixed in a stereotaxic frame with scalp skin retracted, and a hole drilled through the skull plate. The glass needle (yellow arrow) loaded with mesh electronics is visible directly above the skull in this image. A flexible flat cable (FFC) is visible at the left of the image supported on a ceramic scaffold. A 0-80 grounding screw (white arrow) was positioned at the posterior side of the skull. (b) Image showing the relocation of the I/O end of the post-injected mesh electronics (red arrow) towards the FFC. (c) Image post-injection into the brain showing the input/output (I/O) region of the mesh electronics unfolded onto the FFC. Inset shows a zoom-in view of the red dashed box, which highlights the unfolded I/O part (red arrow) of the injected mesh electronics. (d) Image representing the unfolded I/O pads of the mesh electronics which were electrically-connected to the FFC using the conductive ink printing process described in ref. 33. The red dashed box highlighted by the inset shows the details of the bonding with the red and cyan arrows indicating the mesh electronics and conductive ink connections, respectively. (e) Image showing the FFC fixed on top of the mouse skull using dental cement. (f) Image of a fully awake but restrained mouse during chronic in vivo brain recording. The white and black arrows highlight the grounding screw and the connector between FFC and external recording setups, respectively. See Online Methods for all experimental details of surgery and injection. The width of FFC in all panels is ~8 mm.

  5. Supplementary Figure 5: Correlation maps of chronic multiplexed brain recording. (202 KB)

    (a,b) Correlation maps of 16-channel local field potential (LFP) recordings at two (a) and four (b) months post-injection. (c,d) Correlation maps of 16-channel extracellular action potential recordings at two (c) and four (d) months post-injection. Colors indicate the correlation coefficient between any two given channels according to the color bar shown on the far right. All the maps were calculated from 2 s long data traces at both time points. See Online Methods for details of correlation coefficient calculations.

  6. Supplementary Figure 6: Long-term stable recording without signal degradation over six months. (324 KB)

    (a) Representative extracellular action potential recordings from the same electrode (Mouse4-ChannelA shown in Fig. 2b) located in somatosensory cortex (CTX) at different time points post-injection. (b) Spike sorting results of the corresponding recordings shown in (a). All of the spikes from the corresponding time points in (a) are included with ca. 50 spikes at each point. (c) Autocorrelation histograms of average waveforms of sorted spikes from 30 1-min segments within a 30-min recording session for Mouse4-ChannelA at 3 months post-injection. (d) Autocorrelation histograms of average waveforms for each of the two identified clusters (color coded in magenta (neuron 1) and green (neuron 2)) for Mouse4-ChannelA at the 8 time points shown in (b). See Online Methods for details of waveform autocorrelation calculations. (e) Time evolution of normalized average peak-to-peak spike amplitudes across all channels with single-unit action potentials recorded (the average was done across 14, 5, 6 and 7 channels for Mouse 1 to 4, respectively) from four mice. The spike amplitude was normalized (value=1.0, gray dashed horizontal lines) against the average peak-to-peak amplitude values between 5 to 26 weeks post-injection for each channel. (f) Average impedance values at 1 kHz across all channels with recorded single-unit action potentials of the four mice shown in (e) plotted as a function of time over the same time period. The shaded areas in (e) and (f) indicate ±1 standard error of the mean (s.e.m.).

  7. Supplementary Figure 7: Immunohistochemical images of horizontal sectioned mouse brains containing the mesh electronics from different times post-injection. (264 KB)

    (a) GFAP stained images of representative horizontal brain slices used for normalized fluorescence intensity plots (Fig. 2e) showing the interfaces between mesh electronics and astrocytes, and (b) iba-1 stained images of representative horizontal slices used for normalized fluorescence intensity plots (Fig. 2e) showing the interfaces between mesh electronics and microglia at 2, 6 and 12 weeks post-injection. All samples were 10 μm-thick. Blue, cyan and purple correspond to mesh electronics, GFAP and iba-1, respectively. Scale bars: 100 μm.

  8. Supplementary Figure 8: Autocorrelation and cross-correlation analyses of average spike waveforms across all recording time points. (91 KB)

    (a) Auto- and cross-correlation histograms for three identified neurons of Mouse2-ChannelA from week 3 to 34 post-injection shown in Fig. 3b. (b) Auto- and cross-correlation histograms for two identified neurons of Mouse2-ChannelB from week 1 to 34 post-injection shown in Supplementary Fig. 10c. (c) Auto- and cross-correlation histograms for two identified neurons of Mouse3-ChannelA from week 1 to 26 post-injection shown in Supplementary Fig. 10d.

  9. Supplementary Figure 9: Refractory periods of neuron firing. (95 KB)

    (a-c) Interspike interval (ISI) histograms of the data shown in Fig. 3c at 26 weeks post-injection but replotted with a bin size of 1 ms. The data show clearly a 2-3 ms refractory period (orange-shaded regions) for neuron 1 (a), neuron 2 (b) and neuron 3 (c).

  10. Supplementary Figure 10: Consistent tracking of the same group of neurons. (406 KB)

    (a,b) Time evolution of representative single-unit spikes of Mouse2-ChannelB (a) and Mouse3-ChannelA (b) shown in Fig. 2 clustered by principal component analysis (PCA) over eight and six months, respectively. In each plot, the x- and y-axis denote the first and second principal component, respectively, and the z-axis indicates post-injection time. (c,d) Time course analysis of average spike waveforms from each PCA cluster shown in (a) from 1 to 34 weeks post-injection (c), and for each PCA cluster shown in (b) from 1 to 26 weeks (d), respectively. (e,f) Time evolution of ISI histograms of each of the 2 neurons identified from the PCA clusters in (a) and (b) from 3 to 34 (e) and 3 to 26 weeks (f), respectively. Bin size: 20 ms. (g,h) Scatter plot with analysis of variance (ANOVA) (n=32 and 26 for (g) and (h), respectively) of the firing parameter, λ, obtained by fitting each ISI distribution profile shown in (e) and (f) to an exponential decay. All plots for Mouse3 are shown up to 26 weeks post-injection instead of 34 weeks shown for Mouse2, because Mouse3 was older than Mouse2 when the initial mesh electronics injection was carried out, and thus exhibited ageing-associated changes (shown in Fig. 5 and Supplementary Fig. 12) at an earlier post-injection time point.

  11. Supplementary Figure 11: Rayleigh Z-test of neuron phase-locking behavior. (63 KB)

    (a-c) Rayleigh Z-test of all recording data for neuron 1 (a), neuron 2 (b) and neuron 3 (c) identified for Mouse2-ChannelA from week 3 to 34 post-injection (n=32). Each trial presents the recording data at a given week. The null hypothesis is: a given neuron does not have phase-locking behavior at a given week. The majority of trials on the right side of the vertical dashed lines suggest a >95% confidence interval (p<0.05) to reject the null hypothesis.

  12. Supplementary Figure 12: Tracking of the same individual neurons during brain ageing. (389 KB)

    (a,b) Time evolution of representative single-unit spikes of Mouse2-ChannelA (a) and Mouse3-ChannelA (b) shown in Fig. 2 clustered by PCA from 35 to 57 weeks of age. In each plot, the x- and y-axes denote the first and second principal components, respectively, and the z-axis indicates the corresponding mouse age. (c,d) Representative 2D (PC1-PC2 plane) plots of the PCA results shown in (a,b) at week 52 and 57 of mouse age. (e,f) Time evolution of ISI histograms of each of the neurons identified from the PCA clusters in (a) and (b) from 35 to 57 weeks of age. Bin size: 20 ms. (g) Impedance values at 1 kHz of the two channels shown in (a-f) plotted as a function of mouse age over the same period. (h) Immunohistochemical images of a 10 μm-thick horizontal CTX brain slice showing the mesh electronics/brain tissue interface at 1-year mouse age. Red, yellow and blue colors correspond to neurofilaments, NeuN and mesh electronics, respectively. Scale bar: 100 μm. (i) Neurofilament (red), NeuN (yellow), GFAP (cyan) and iba-1 (purple) fluorescence intensity normalized against background values (gray dashed horizontal lines) plotted versus distance from the interface (see Online Methods). The pink-shaded regions indicate the interior of the mesh electronics. All error bars in this figure reflect ±1 standard error of the mean (s.e.m.).

  13. Supplementary Figure 13: Spike recordings from a freely behaving mouse. (273 KB)

    (a) Sorted single-unit action potentials from chronic freely behaving mouse recordings. Each column represents the sorted spikes from an individual channel shown in Fig. 6 at 5, 10 and 27 weeks post-injection. (b) Noise distributions of all red unit clusters of Channels A-E shown at week 10 in a (I), and the intrinsic recording noise of each corresponding electrode of the channel at the same week (II). See Online Methods for details of noise calculations. (c) Single-unit spikes of Channel D in (a) clustered by PCA when the mouse was actively whisking (I) and foraging (II) at week 5, 10 and 27.

  14. Supplementary Figure 14: Autocorrelation analyses of average waveforms from week 1 to 8 post-injection. (89 KB)

    (a) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse2-ChannelA from weeks 1 to 8 post-injection shown in Fig. 3b. (b) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse2-ChannelB from weeks 1 to 8 shown in Supplementary Fig. 10c. Inset: Cross-correlation histogram of all raw spikes for neuron 2 between weeks 1 and 8 (light green, dashed line) and autocorrelation histogram of all raw spikes for neuron 2 on week 8 only (dark green, solid line). (c) Autocorrelation histograms of average waveforms for neurons 1 (magenta) and 2 (green) for Mouse3-ChannelA from weeks 1 to 8 shown in Supplementary Fig. 10d. See Online Methods for details of autocorrelation calculations.

Video

  1. Video 1: Chronic recordings from a freely behaving mouse with implanted mesh electronics probe. (4.4 MB, Download)
    This video shows a mouse with a head-mounted voltage-amplifier roaming freely in a cage with randomly positioned food pellets during recording. The frame rate is 25 frames per second (fps) and the video is played at 1× real time. The mesh electronics was injected into the somatosensory cortex and hippocampus, and bonded to the interface cable as described in the Online Methods.

PDF files

  1. Supplementary Text and Figures (2,381 KB)

    Supplementary Figures 1–14 and Supplementary Tables 1–3

Additional data