A high-density, high-channel count, multiplexed μECoG array for auditory-cortex recordings

Abstract

Our understanding of the large-scale population dynamics of neural activity is limited, in part, by our inability to record simultaneously from large regions of the cortex. Here, we validated the use of a large-scale active microelectrode array that simultaneously records 196 multiplexed micro-electrocortigraphical (μECoG) signals from the cortical surface at a very high density (1,600 electrodes/cm(2)). We compared μECoG measurements in auditory cortex using a custom "active" electrode array to those recorded using a conventional "passive" μECoG array. Both of these array responses were also compared with data recorded via intrinsic optical imaging, which is a standard methodology for recording sound-evoked cortical activity. Custom active μECoG arrays generated more veridical representations of the tonotopic organization of the auditory cortex than current commercially available passive μECoG arrays. Furthermore, the cortical representation could be measured efficiently with the active arrays, requiring as little as 13.5 s of neural data acquisition. Next, we generated spectrotemporal receptive fields from the recorded neural activity on the active μECoG array and identified functional organizational principles comparable to those observed using intrinsic metabolic imaging and single-neuron recordings. This new electrode array technology has the potential for large-scale, temporally precise monitoring and mapping of the cortex, without the use of invasive penetrating electrodes.

Keywords: auditory cortex; electrocorticography; tonotopy; topography; μECoG.

Fig. 1.

Micro-electrocortigraphical (μECoG) recording array and technical details of electrode array fabrication. A: microscope images of the fabrication process at each layer. Top left: ”U“-shaped silicon nanoribbons transfer-printed to the flexible polyimide substrate are shown. Top middle: the first metal layer provided horizontal row select signals, connections to upper layers for the electrode input, and source/drain contacts. Top right: the second metal layer provided connections for the common power wire and shared column output line. Bottom left and bottom middle: subsequent metal layers and encapsulation layers provided connection between the surface electrodes and the load transistor (Tr) as well as protection for the active circuit elements from biological fluids. Bottom middle: lastly, a final encapsulation layer and Pt electrode deposition step finalizes the device. Bottom right: schematic circuit diagram of a single unit-cell containing two matched Tr. B: the 14 × 14 organization of the electrodes in the μECoG recording array. The inset shows a magnified view of a 3 × 3 region of the array. C: the recording array on the lateral surface of a rat's brain is shown. Pt, platinum.

Fig. 2.

Topographic distribution of frequency response areas (FRAs) derived from μECoG activity. A1: topographic organization of the FRAs generated from each recording site on the array. Each square demarcates the relative location of each electrode and the FRA generated from the neural activity recorded at that site. For each FRA, frequency in kHz is plotted on the x-axis, and sound pressure level (SPL; dB) is plotted on the y-axis. Red regions indicate frequency-sound level combinations that elicited neural activity that was above baseline activity; see color bar. Note that all of the FRAs are normalized relative to the site with the maximum response using a common scale as the number of SDs above the baseline voltage of each recording site. Blue regions indicate frequency-sound level combinations that elicited neural activity that was at or near the baseline. A, B, and C: three FRAs were selected to illustrate the variability in tuning properties observed with the μECoG array. The respective panel letter denotes the topographic location of each of these FRA within the μECoG array (A1). SD, standard deviation.

Fig. 3.

Reliability of single-trial and trial-averaged μECoG responses from a single electrode site on the active μECoG array. A: single-trial μECoG voltage response traces to single tone pips at each frequency (x-axis) and sound level (y-axis) combination. B: trial-averaged μECoG voltage response traces that were generated from six tone pip presentations at each level and frequency combination. Like A, these data are organized as a function of frequency (x-axis) and sound level (y-axis). C and D: the FRAs generated from the single-trial and trial-averaged μECoG responses, respectively. The color bar indicates measured peak-to-peak voltage from the recorded voltage traces in A and B.

Fig. 4.

High correspondence between the best frequencies (BFs) of single-trial FRAs and trial-averaged FRAs. Three examples of single-trial FRAs (A–C) are shown with their corresponding trial-averaged FRAs (D–F). For these plots, color indicates the maximum peak-to-peak voltage: blue indicates 0 V, and dark red indicates the highest peak-to-peak voltage observed for that particular electrode site. A map of BF that was generated from the single-trial FRA data (trial number 3) at each electrode site is shown in G, whereas the trial-averaged map of BF is shown in H. The color bar between G and H indicates the frequency scale (kHz) of these two BF maps. I: the site-by-site correlation (r = 0.95 ± 0.01, mean ± SE) between the single-trial BF and the trial-averaged BF for significant FRAs obtained from this animal; the gray line is the line of unity. Data from this same animal are shown in Fig. 8.

Fig. 5.

Correlation between FRAs generated from intrinsic optical imaging (IOI) and custom active-μECoG-array recordings. A: composite image of the lateral surface of a rat's brain, including surface vasculature and IOI responses to tone sequences; the color bar indicates the tone BF. The gray and black dots indicate the locations of each electrode of the array: black dots indicate those electrodes that overlapped with the primary auditory cortex plus the area dorsal to primary (A1) (labeled A1) and ventral auditory field (VAF), as defined by IOI. Scale bars indicate 500 μm, dorsal (D) and ventral (V) anatomic axes. B and C: the BF maps generated from IOI and μECoG FRA data, respectively. The IOI BF was averaged over a spatial region corresponding to the spacing (i.e., 250 × 250 μm) between each recording site of the active μECoG array. The color bars indicate the BF (kHz) of these maps. D: a significant (results) site-by-site correlation between the BF values generated from the μECoG FRA and the IOI is observed. The dotted line is the line of unity. The white areas in B and C either were recording sites that were not in the area of interest or were sites that did not have statistically reliable BFs. The data shown in this figure are the results from a single animal. Spectrotemporal response field (STRF) and STRF BF data for this same animal are shown in Fig. 10.

Fig. 6.

Topographic distribution of FRAs derived from commercially available passive μECoG recordings. A: the lateral surface of a rat's brain, including surface vasculature and the passive NeuroNexus array positioned on the surface of the rat's brain. Scale bars indicate 500 μm, D and R (rostral) indicate the anatomic axes. The FRAs generated from this array are shown in B. Red regions indicate frequency-sound level combinations that elicited neural activity that was above baseline; see color bar. Blue regions indicate frequency-sound level combinations that elicited neural activity that was at baseline. Each FRA is normalized relative to the voltage values recorded at that electrode site. The FRAs are organized relative to the spatial position of each recording site on the array. The data shown in this figure are the results from a single animal.

Fig. 7.

Correlation between FRAs obtained from IOI and conventional passive μECoG recording. A: composite image of the lateral surface of a rat's brain, including surface vasculature and IOI response to tone sequences; the color bar indicates the tone BF. The gray and white dots indicate the locations of each electrode of the array: black dots indicate those electrodes that overlapped with the primary auditory cortex and the area dorsal to A1 (all labeled A1) and VAF, as defined by the IOI. B and C: the BF maps generated from IOI and μECoG FRA data, respectively. The optical BF was averaged over a spatial region corresponding to the spacing (i.e., 300 × 300 μm) between each recording site of the passive μECoG array. The color bar in B indicates the BF (kHz). D: a significant (results) site-by-site correlation between the BF values generated from the conventional passive μECoG FRA and the IOI. The diagonal line is the line of unity for comparison. The white areas in B and C either were recording sites that were not in the area of interest, or were sites that did not have statistically reliable BFs. The data shown in this figure are the results from a single animal.

Fig. 8.

Topographic organization and relationship between BF and bandwidth (BW) measured with the custom μECoG array. A1 and A2: position maps for tone BF and BW, respectively, derived from the FRA obtained at a sound level of 65 dB SPL. Scale bars indicate 500 μm, D and V anatomic axes. Color bars indicate range of values in all corresponding maps. A3: an inverted “U-shaped” relationship is observed between BF vs. BW measured at each cortical site. B1–B3 and C1–C3 follow the same convention as in A1–A3; however, data are obtained at sound levels of 75 and 85 dB SPL, respectively. The data shown in this figure are the results from a single animal, same animal as in Fig. 4.

Fig. 9.

A comparison of the FRA BW selectivity between custom active and conventional passive μECoG arrays. The median BW for each array is plotted as a function of sound level. Error bars indicate the standard error obtained via bootstrapping across measurements (materials and methods). *Sound levels in which the BWs of custom vs. conventional array responses were significantly different (P < 0.001; rank-sum test).

Fig. 10.

Topographic distribution of STRFs and BFs derived from custom active-μECoG-array responses. A: the topographic organization of STRFs that were generated from each recording site on the array. Each square demarcates the relative location of each electrode and the STRF that was generated from the neural activity recorded at that site. For each STRF, delay in milliseconds is plotted on the x-axis, and frequency in octaves is plotted on the y-axis. Blue regions indicate excitatory voltage responses were temporally phase-locked with the auditory stimulus, whereas blue values indicate suppressive/inhibitory responses (see Fig. 11 for typical STRF patterns). The color scale on all STRFs is normalized relative to the recording site with the strongest response. The reliability of each STRF is shown in B for each recording site. The value of each STRFs reliability is indicated by the color bar: STRFs with lowest reliability are blue, and those with highest reliabilities are dark red. C: the topographic organization of BF. The BF from each recording site was calculated from the STRFs shown in A. The BF of each site is indicated by the color bar: sites with ∼1-kHz BFs are dark blue, and those with ∼32-kHz BFs are dark red. The data shown in this figure are the results from a single animal. The pure-tone BF responses and various STRF response parameters for this animal are shown in Figs. 5 and 12, respectively.

Fig. 11.

Examples of STRFs from the primary auditory cortex. A–F: each panel shows a STRF generated from a different recording site. As indicated by the color bar, increasingly red regions indicate spectrotemporal combinations in which the μECoG signal was increasing, whereas increasingly blue regions indicate spectrotemporal combinations in which the μECoG signal was at decreasing. For both, increases and decreases were temporally phase-locked with the auditory stimulus. The STRFs have been normalized relative to the maximum values.

Fig. 12.

Topographic organization of STRF parameters. A: the organization of STRF BW in units of octaves. B: the organization of the best spectral modulation in units of Hertz/octave. C: the organization of response delay in units of milliseconds. D: the organization of the best temporal modulation in Hertz. The color bar next to each panel indicates the respective range of values. See materials and methods for details on how these parameters were calculated. In each panel, the thick black line delineates the borders of the primary auditory cortex and the VAF. The spatial orientation of the data in B–D is the same as that shown in A. The data shown in this figure are the results from a single animal. The boundaries between the primary auditory cortex and the VAF were determined by the direction of the BF gradients in the optical image, as described previously (Higgins et al. 2010).

Fig. 13.

Correlation between STRF parameters. The correlations between different A–F: STRF parameters are shown in each of the panels. Each symbol in each graph represents the data value (e.g., BF and BW for A) generated from the STRF at a single recording site. The different symbols (circle, triangle, and cross) represent data from different animals.

Fig. 14.

The pairwise correlation between recording sites was dependent on their spatial separation and the difference between the STRF BFs for each site. A: the median cross-correlation functions as a function of the spatial separation between recording sites. B: the median cross-correlation functions as a function of the BF difference. C: the interdependence between spatial separation and BF difference. In each plot, the spatial separation is held constant, whereas the cross-correlation functions are plotted as a function of BF difference. See B for the color code of BF difference. For all of the panels, each cross-correlation function represents the median function that was calculated from the subset of recording sites that had significant STRFs for each combination of spatial separation and BF difference. The data shown in this figure are the results from a single animal.